CN113336748B

CN113336748B - GPX4 protein degradation agent, preparation method and application thereof, and anti-tumor cell drug

Info

Publication number: CN113336748B
Application number: CN202110391225.4A
Authority: CN
Inventors: 徐萍; 王超; 郑藏鑫; 孙丹; 许凤荣
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2021-04-12
Filing date: 2021-04-12
Publication date: 2022-03-25
Anticipated expiration: 2041-04-12
Also published as: CN113336748A

Abstract

The invention provides a GPX4 protein degradation agent, a preparation method thereof and an anti-tumor cell drug, belonging to the technical field of drug application. The GPX4 protein degrading agent provided by the invention has a protein degradation targeting chimera (PROTAC) molecular structure, a mother nucleus structure is used as a small molecular ligand combined with target protein, an A2 substituent is used as a small molecular ligand combined with an E3 ubiquitin ligase compound, an A1 substituent is used as a connecting group for connecting two ligands, and the GPX4 protein degrading agent with the structure can specifically recognize GPX4 protein and effectively ubiquitinate and degrade the GPX4 protein, so that the death of tumor cells is induced.

Description

GPX4 protein degradation agent, preparation method and application thereof, and anti-tumor cell drug

Technical Field

The invention relates to the technical field of medicine application, in particular to a GPX4 protein degradation agent, a preparation method and application thereof, and an anti-tumor cell medicine.

Background

Stockwell et al, university of columbia, 2012 proposed the concept of "iron death" (ferrotosis), an iron-dependent, programmed cell death pattern characterized by the accumulation of intracellular reactive oxygen species. Iron death is often caused by a defect in the intracellular lipid hydroperoxide scavenging system, resulting in the accumulation of large amounts of lipid hydroperoxides, which ultimately cause cell damage and death.

During the biological evolution process, cells develop a defense mechanism against iron death by oxidative lipid accumulation, known as the System Xc-GSH-GPX 4 pathway. Glutathione peroxidase 4(GPX4) is a core protein in the pathway, is one of 8 subtypes of the glutathione peroxidase family, is an important superoxide dismutase, and has a Selenocysteine (Sec) as the catalytic active center. GPX4, which usually uses GSH as a cofactor, can convert intracellular lipid hydroperoxides into nontoxic aliphatic alcohols and also catalyze the reduction of other organic peroxides such as hydrogen peroxide, thereby protecting cells from oxidative stress and inhibiting the occurrence of iron death. Therefore, inhibition of GPX4 activity affects the ability of GPX4 to scavenge lipid peroxides, ultimately leading to the occurrence of cellular iron death.

Because the GPX4 protein lacks a traditional drug pocket, the design of a compound targeting GPX4 has certain difficulty, but a small molecule covalent inhibitor can still damage the enzymatic activity of the GPX4 by irreversible combination with a selenocysteine residue at the active center of the GPX 4. Therefore, the GPX4 inhibitor is mainly based on covalent inhibitor at present. The GPX4 inhibitors that have been reported are mainly represented by the following formula:

at present, no GPX4 inhibitor entering the clinical research stage is reported, and the inhibitor also has the problems of off-target effect, bioavailability and the like.

Disclosure of Invention

In view of the above, the invention aims to provide a GPX4 protein degradation agent, a preparation method and an application thereof, and an anti-tumor cell drug, and the GPX4 protein degradation agent provided by the invention has a PROTAC molecular structure, and can effectively degrade GPX4 protein, so as to induce tumor cell iron death.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a GPX4 protein degrading agent, which has a structure shown in formula 1:

in the formula 1, A₁Is of the formula A₁₁～A₁₃Any one of the substituents shown:

A₁₁and A₁₂M is 1 to 8, A₁₃Wherein n is 5 to 20, o is 0 to 3, and Z is CO or CH₂；

A₂Is of the formula A₂₁～A₂₂Any one of the substituents shown:

A₂₁wherein X is CH₂Or CO; a. the₂₂In which Y is CH₃Or H.

Preferably, the GPX4 protein degradation agent has a structure represented by formula 2, formula 3, formula 4, formula 5, formula 6, or formula 7:

in the formula 2, m is 1-8;

in the formula 3, m is 1-8;

in the formula 4, m is 1-8;

in the formula 5, n is 5-20, o is 0-3, and Z is CO;

in the formula 6, n is 5-20, o is 0-3, and Z is CO;

in the formula 7, n is 5-20, o is 0-3, and Z is CO;

in the formula 8, n is 5-20, o is 0-3, and Z is CH₂；

The invention provides a preparation method of the GPX4 protein degradation agent, which comprises the following steps:

when A is₁Is A₁₁Or A₁₂The preparation method of the GPX4 protein degradation agent comprises the following steps:

carrying out Click reaction on an E3 ligand compound substituted by an azido polyethylene glycol chain and a first GPX4 ligand to obtain a GPX4 protein degradation agent;

the azido polyethylene glycol chain-substituted E3 ligand compound has a structure shown as a formula A-1, A-2 or A-3:

the first GPX4 ligand has the structure shown in formula B:

when A₁Is A₁₃The preparation method of the GPX4 protein degradation agent comprises the following steps:

condensing E3 ligand compound substituted by piperazine ring carbon chain with a second GPX4 ligand to obtain GPX4 protein degradation agent;

or the E3 ligand compound substituted by the piperazine ring carbon chain and the third GPX4 ligand are subjected to nucleophilic substitution reaction to obtain the GPX4 protein degradation agent;

the piperazine ring carbon chain substituted E3 ligand compound has a structure shown in a formula C-1, a formula C-2, a formula C-3 or a formula C-4:

the second GPX4 ligand has the structure shown in formula D:

the third GPX4 ligand has the structure shown in formula E:

preferably, the preparation method of the azido polyethylene glycol chain-substituted E3 ligand compound with the structure shown in the formula A-1 comprises the following steps:

carrying out substitution reaction on a polyethylene glycol compound with a structure shown in a formula a and lenalidomide with a structure shown in a formula b to obtain an azido polyethylene glycol chain heterocyclic compound with a structure shown in a formula A-1;

the preparation method of the azido polyethylene glycol chain-substituted E3 ligand compound with the structure shown in the formula A-2 comprises the following steps:

carrying out substitution reaction on a polyethylene glycol compound with a structure shown in a formula c and a compound with a structure shown in a formula d to obtain an azido-polyalcohol chain heterocyclic compound with a structure shown in a formula A-2;

the preparation method of the azido polyethylene glycol chain-substituted E3 ligand compound with the structure shown as A-3 comprises the following steps:

carrying out condensation reaction on a polyethylene glycol compound with a structure shown as a formula e and a compound with a structure shown as a formula f to obtain an azido-polyalcohol chain heterocyclic compound with a structure shown as a formula A-3;

preferably, the preparation method of the first GPX4 ligand having the structure shown in formula B comprises the following steps:

carrying out substitution reaction on a compound with a structure shown in a formula i and chloroacetyl chloride to obtain a first GPX4 ligand with a structure shown in a formula B;

preferably, the preparation method of the piperazine ring carbon chain substituted E3 ligand compound with the structure shown in formula C-1 comprises the following steps:

carrying out substitution reaction on bromoalkanol and 1-Boc-piperazine to obtain a compound with a structure shown in a formula j;

carrying out substitution reaction on a compound with a structure shown as a formula j and p-toluenesulfonyl chloride to obtain a compound with a structure shown as a formula k;

carrying out substitution reaction on a compound with a structure shown as a formula k and lenalidomide to obtain a compound with a structure shown as a formula L;

carrying out deprotection reaction on the compound with the structure shown in the formula L to obtain an E3 ligand compound containing piperazine ring carbon chain substitution with the structure shown in the formula C-1;

the preparation method of the piperazine ring carbon chain substituted E3 ligand compound with the structure shown as C-2 comprises the following steps:

carrying out substitution reaction on a compound with a structure shown in a formula k and a compound with a structure shown in a formula f to obtain a compound with a structure shown in a formula m;

carrying out deprotection reaction on the compound with the structure shown in the formula m to obtain an E3 ligand compound containing piperazine ring carbon chain substitution with the structure shown in the formula C-2;

the preparation method of the piperazine ring carbon chain substituted E3 ligand compound with the structure shown as C-3 comprises the following steps:

carrying out substitution reaction on a compound with a structure shown as a formula k and azido trimethyl silane to obtain a compound with a structure shown as a formula n:

a compound having a structure represented by formula n and H₂Carrying out hydrogenation reaction to obtain a compound with a structure shown as a formula o;

carrying out substitution reaction on the compound with the structure shown in the formula o and the compound with the structure shown in the formula d to obtain a compound with the structure shown in the formula p;

carrying out deprotection reaction on the compound with the structure shown in the formula p to obtain an E3 ligand compound containing piperazine ring carbon chain substitution and having the structure shown in C-3;

the preparation method of the piperazine ring carbon chain substituted E3 ligand compound with the structure shown as C-4 comprises the following steps:

carrying out substitution reaction on a compound with a structure shown as a formula C-2 and 1-Boc-4-bromo-piperidine to obtain a compound with a structure shown as a formula w;

and carrying out deprotection reaction on the compound with the structure shown in the formula w to obtain the E3 ligand compound containing piperazine ring carbon chain substitution with the structure shown in the formula C-4.

Preferably, the preparation method of the second GPX4 ligand having the structure shown in formula D comprises the following steps:

carrying out substitution reaction on the compound with the structure shown as the formula t and chloroacetyl chloride to obtain a second GPX4 ligand with the structure shown as the formula D;

preferably, the preparation method of the third GPX4 ligand having the structure shown in formula E comprises the following steps:

carrying out substitution reaction on a compound with a structure shown as a formula u and chloroacetyl chloride to obtain a third GPX4 ligand with a structure shown as a formula E;

the invention provides application of the GPX4 protein degradation agent in preparation of anti-tumor drugs and drug-resistant tumor drugs.

The invention provides an anti-tumor medicament, which comprises a medicament active component and a medicament auxiliary material; the active pharmaceutical ingredient is the GPX4 protein degradation agent.

The invention provides a GPX4 protein degradation agent which has a structure shown in a formula 1. The GPX4 protein degrading agent provided by the invention has a protein degradation targeting chimera (PROTAC) molecular structure, a mother nucleus structure of the GPX4 protein degrading agent is used as a small molecular ligand combined with a target protein, A₂The substituent serves as a small molecule ligand for binding E3 ubiquitin ligase complex, A₁The substituent is used as a connecting group for connecting two ligands, and the GPX4 protein degrading agent with the structure can specifically recognize GPX4 protein and effectively ubiquitinate GPX4 protein, so that tumor cell iron death is induced. The evaluation of cell level and activity in animal body shows that the GPX4 protein degrading agent provided by the invention can effectively degrade GPX4 protein in cells and mouse tumor tissues, and can induce tumor cell iron death and inhibit mouse tumor growth; meanwhile, the GPX4 protein degrading agent provided by the invention has good biological activity, can rapidly and efficiently induce ubiquitination degradation of GPX4 protein in human fibrosarcoma cells (HT1080) at a nanomolar level, and also has good GPX4 degradation effect and anti-proliferation effect on human lung cancer cells (Calu-1), breast cancer cells (MCF7), wild type and drug-resistant human non-small cell lung cancer cells (H1650).

Drawings

FIG. 1 shows the result of GPX4 degradation with PEG Linker type degradation agent;

FIG. 2 shows the result of GPX4 protein degradation by carbon chain type degradation agent;

FIG. 3 shows that the effect of the degradation agent on the degradation of GPX4 protein is concentration-dependent and time-dependent;

FIG. 4 shows that the degradation agent R27 degrades GPX4 protein through ubiquitin-proteasome pathway;

FIG. 5 shows tumor cell proliferation inhibition experiments;

FIG. 6 is an iron death mechanism study experiment;

FIG. 7 shows that the degradation of GPX4 protein in drug-resistant tumor cells induced by degradation agent and the inhibition of tumor cell proliferation;

FIG. 8 shows the degradation of GPX4 protein in tumor tissue by R27;

FIG. 9 is a graph of the effect of R27 on mouse tumor volume and body weight.

Detailed Description

in formula 1, A1 is formula A₁₁～A₁₃Any one of the substituents shown:

A₂Is of the formula A₂₁～A₂₂Any one of the substituents shown:

A₂₁wherein X is CH₂Or CO; a. the₂₂In which Y is CH₃Or H;

a is described₁Left end connecting key and A₂The right end of the connecting key is connected with the connecting key.

As a preferred embodiment of the present invention, the GPX4 protein degradation agent has a structure represented by formula 2, formula 3, formula 4, formula 5, formula 6, or formula 7:

in the formula 2, m is 1-8;

in the formula 3, m is 1-8;

in the formula 4, m is 1-8;

in the formula 5, n is 5-20, o is 0-3, and Z is CO;

in the formula 6, n is 5-20, o is 0-3, and Z is CO;

in the formula 7, n is 5-20, o is 0-3, and Z is CO;

in the formula 8, n is 5-20, o is 0-3, and Z is CH₂。

Traditional small molecules block the function of proteins by directly binding with active sites of target proteins, and protein degradation targeting chimera (PROTAC) technology utilizes the protein degradation mechanism of cells to remove specific proteins from the cells, which is an alternative method for targeted therapy. The PROTAC molecule includes three moieties: a small molecule ligand binding to the target protein, a small molecule ligand binding to the E3 ubiquitin ligase complex, and a structurally stable linker linking the two ligands. The bifunctional molecule can draw E3 ubiquitin ligase to the target protein, so that the target protein is effectively ubiquitinated, the ubiquitinated target protein is further degraded by proteasome, and the PROTAC molecule can continue to degrade new target protein.

The GPX4 protein degradation agent provided by the invention has a protein degradation targeting chimera (PROTAC) molecular structure and a mother nucleus structure

As a small molecule ligand for binding target protein, the A2 substituent is used as a small molecule ligand for binding E3 ubiquitin ligase complex, the A1 substituent is used as a connecting group for connecting two ligands, and the GPX4 protein degradation agent with the structure can specifically recognize GPX4 protein and effectively ubiquitinate GPX4 protein, so that tumor cell iron death is induced. The evaluation of cell level and activity in animal body shows that the GPX4 protein degrading agent provided by the invention can effectively degrade GPX4 protein in cells and mouse tumor tissues, and can induce tumor cell iron death and inhibit mouse tumor growth; meanwhile, the GPX4 protein degrading agent provided by the invention has good biological activity, can rapidly and efficiently induce ubiquitination degradation of GPX4 protein in human fibrosarcoma cells (HT1080) at a nanomolar level, and also has good GPX4 degrading effects on human lung cancer cells (Calu-1), breast cancer cells (MCF7), wild type and drug-resistant human non-small cell lung cancer cells (H1650).

when A is₁Is composed of

When the temperature of the water is higher than the set temperature,

the preparation method of the GPX4 protein degradation agent comprises the following steps:

the azido polyalcohol chain heterocyclic compound has a structure shown as a formula A-1, A-2 or A-3:

the first GPX4 ligand has the structure shown in formula B:

in the present invention, the mole ratio of the azido polyethylene glycol chain-substituted E3 ligand compound to the first GPX4 ligand is preferably 0.11: 0.12; in the present invention, the catalyst in the ring formation reaction is preferably sodium ascorbate and copper sulfate, and the molar ratio of the sodium ascorbate, the copper sulfate and the azido polyalcohol chain heterocyclic compound is preferably 0.26: 0.13: 0.11. In the present invention, the solvent used in the click reaction is preferably N, N-dimethylformamide.

In the invention, the temperature of the cyclization reaction is preferably 25-100 ℃, more preferably the time is preferably 1-50 h, and more preferably 5-20 h.

After the cyclization reaction, the invention preferably carries out post-treatment on the obtained cyclization reaction liquid; the post-treatment preferably comprises the steps of:

and quenching the cyclization reaction solution by using water, and sequentially performing extraction, washing, drying and column separation to obtain a pure GPX4 protein degradation agent.

In the present invention, the extractant for extraction is preferably ethyl acetate; the washing detergent is preferably a saturated NaCl solution; the drying agent is preferably anhydrous Na₂SO₄(ii) a The stationary phase of column chromatography separation is preferably 200-300 mesh silica gel, the mobile phase is preferably dichloromethane and methanol, and the mobile phase is preferably dichloromethane and methanolThe volume ratio of (A) to (B) is preferably 500-1000: 1.

In the present invention, the method for preparing the azido polyethylene glycol chain-substituted E3 ligand compound having the structure represented by the formula a-1 preferably comprises the steps of:

carrying out substitution reaction on a polyalcohol compound with a structure shown as a formula a and lenalidomide with a structure shown as a formula b to obtain an azido polyethylene glycol chain-substituted E3 ligand compound with a structure shown as a formula A-1;

in the present invention, the molar ratio of the polyol compound to lenalidomide is preferably 1: 1; in the present invention, the catalyst used for the substitution reaction is preferably K₂CO₃And KI; said K₂CO₃The molar ratio of KI to lenalidomide is preferably 3.24: 0.49: 1.62. In the present invention, the solvent used for the substitution reaction is preferably acetonitrile. In the invention, the temperature of the substitution reaction is preferably the reflux temperature, and the time is preferably 1-50 h, and more preferably 5-20 h.

After the substitution reaction, the invention preferably carries out post-treatment on the obtained substitution reaction liquid; in the present invention, the post-treatment preferably comprises the steps of:

and sequentially filtering the substitution reaction liquid, extracting an organic phase, washing and drying to obtain the pure azido polyethylene glycol chain-substituted E3 ligand compound with the structure shown in the formula A-1.

The present invention does not require any particular filtration means, and filtration means known to those skilled in the art may be used. In the invention, the extracting agent for extraction is ethyl acetate and water; the washing detergent is preferably a saturated NaCl solution; the drying agent is preferably anhydrous Na₂SO₄。

In the present invention, the method for producing an azido polyethylene glycol chain-substituted E3 ligand compound having a structure represented by the formula A-2 preferably comprises the steps of:

carrying out substitution reaction on a polyalcohol compound with a structure shown in a formula c and a compound with a structure shown in a formula d to obtain an azido polyethylene glycol chain substituted E3 ligand compound with a structure shown in a formula A-2;

in the present invention, the molar ratio of the polyol compound to the compound having the structure represented by formula d is preferably 1.91: 1.59. In the present invention, the solvent used for the substitution reaction is preferably N, N-diisopropylethylamine. In the invention, the temperature of the substitution reaction is preferably 90 ℃, and the time is preferably 8-12 h.

and sequentially extracting, washing an organic phase, drying and separating by column chromatography to obtain the pure azido polyethylene glycol chain-substituted E3 ligand compound with the structure shown in the formula A-2.

In the present invention, the extraction solvent is preferably ethyl acetate, and the number of times of extraction is preferably 3. In the present invention, the washing detergent is preferably a saturated NaCl solution; the drying agent is preferably anhydrous Na₂SO₄. The stationary phase of column chromatography separation is preferably 200-300 mesh silica gel, the mobile phase is preferably dichloromethane and methanol, and the volume ratio of the mobile phase is preferably 500-1000: 1.

In the present invention, the method for producing the azido-polyalcohol chain heterocyclic compound having a structure represented by A-3 preferably comprises the steps of:

carrying out condensation reaction on a polyalcohol compound with a structure shown in a formula E and a compound with a structure shown in a formula f to obtain an azido polyethylene glycol chain substituted E3 ligand compound with a structure shown in a formula A-3;

in the present invention, the molar ratio of the polyol compound to the compound having the structure represented by formula f is preferably 0.3: 0.16. In the present invention, the condensation reagent used in the condensation reaction is preferably HATU, and the solvent is preferably N, N-diisopropylethylamine. In the invention, the condensation reaction is preferably carried out at room temperature, and the time is preferably 1-20 h, and more preferably 5-10 h.

After the condensation reaction, the invention preferably carries out post-treatment on the obtained condensation reaction liquid; in the present invention, the post-treatment preferably comprises the steps of:

and sequentially extracting, washing an organic phase, drying and separating by column chromatography to obtain the pure azido polyethylene glycol chain-substituted E3 ligand compound with the structure shown by A-3.

In the present invention, the extraction solvent is preferably ethyl acetate, and the number of times of extraction is preferably 3. In the present invention, the washing detergent is preferably 5 wt% citric acid solution, saturated NaHCO solution, in that order₃Solution and saturated NaCl solution; in the present invention, the drying agent is preferably anhydrous Na₂SO₄. The stationary phase of column chromatography separation is preferably 200-300 mesh silica gel, the mobile phase is preferably dichloromethane and methanol, and the volume ratio of the mobile phase is preferably 500-1000: 1.

In the present invention, the preparation method of the first GPX4 ligand having the structure shown in formula B comprises the following steps:

in the invention, the molar ratio of the compound with the structure shown in the formula i to chloroacetyl chloride is preferably 0.8: 0.96, the temperature condition of the substitution reaction is preferably ice bath, and the time is preferably 1-20 h, and more preferably 7 h.

In the present invention, the preparation method of the compound having the structure represented by formula i preferably comprises the following steps:

carrying out condensation reaction on p-formylbenzoic acid and propiolic acid to obtain a compound with a structure shown in a formula g:

in the present invention, the molar ratio of p-formylbenzoic acid to propiolic acid is preferably 53.29: 79.93; the solvent for the condensation reaction is preferably THF. In the present invention, the condensation reaction is preferably carried out at room temperature for 14 hours.

Carrying out cyclization reaction on a compound with a structure shown in a formula g and a compound with a structure shown in a formula h to obtain a compound with a structure shown in a formula i;

in the present invention, the molar ratio of the compound having the structure represented by formula g to the compound having the structure represented by formula h is preferably 10.68: 11.75; in the invention, the temperature of the cyclization reaction is preferably the reflux temperature, and the time is preferably 8-12 h.

When A₁Is composed of

carrying out condensation reaction on an E3 ligand compound containing piperazine ring carbon chain substitution and a second ligand or carrying out nucleophilic substitution reaction on a third ligand to obtain a GPX4 protein degrading agent;

the piperazine ring carbon chain substituted E3 ligand compound has a structure shown in a formula C-1, a formula C-2 or a formula C-3:

the second GPX4 ligand has the structure shown in formula D:

in the present invention, the molar ratio of the piperazine ring carbon chain-containing heterocyclic compound to the second ligand is preferably 1: 1; in the present invention, the condensation reagent used in the condensation reaction is preferably HATU, and the solvent is preferably N, N-diisopropylethylamine. In the present invention, the condensation reaction is preferably carried out at room temperature for 5 hours. In the present invention, the condensation reaction is preferably carried out at room temperature for 2 hours.

After the condensation reaction is finished, the invention preferably carries out post-treatment on the obtained condensation reaction liquid; in the present invention, the post-treatment preferably comprises the steps of:

and sequentially extracting, washing an organic phase, drying and separating by column chromatography to obtain a pure GPX4 protein degradation agent.

In the present invention, the extractant for extraction is preferably water and ethyl acetate; the washing detergent is preferably a saturated NaCl solution; in the present invention, the drying agent is preferably anhydrous Na₂SO₄. The stationary phase of column chromatography separation is preferably 200-300 mesh silica gel, the mobile phase is preferably dichloromethane and methanol, and the volume ratio of the mobile phase is preferably 500-1000: 1.

The third GPX4 ligand has the structure shown in formula E:

in the invention, the molar ratio of the piperazine ring carbon chain heterocyclic compound to the third ligand is preferably 1: 1; in the present invention, the reagent for nucleophilic substitution reaction is preferably K₂CO₃And KI, the solvent is preferably acetonitrile. In the invention, the reaction temperature is preferably 50-150 ℃, and the reaction time is preferably 2-10 h. In the invention, the temperature of the nucleophilic substitution reaction is preferably 50-150 ℃, and the time is preferably 2-10 h.

After the substitution reaction is finished, the invention preferably carries out post-treatment on the obtained condensation reaction liquid; in the present invention, the post-treatment preferably comprises the steps of:

In the present invention, the method for preparing the piperazine ring carbon chain-containing heterocyclic compound having the structure represented by the formula C-1 preferably comprises the steps of:

in the present invention, the molar ratio of the bromoalkanol to 1-Boc-piperazine is preferably 2.95: 2.68; in the present invention, the catalyst used for the substitution reaction is preferably K₂CO₃And KI; the solvent used for the substitution reaction is preferably acetonitrile. In the present invention, the temperature of the substitution reaction is preferably a reflux temperature, and the time is preferably 2 hours.

and (3) sequentially extracting, washing an organic phase, drying and separating by a chromatographic column to obtain a pure compound with the structure shown in the formula j.

in the invention, the mol ratio of the compound with the structure shown in the formula j to the p-methylbenzenesulfonyl chloride is preferably 3.66: 5.49; in the invention, the temperature of the substitution reaction is preferably room temperature, and the time is preferably 8-12 h.

in the invention, the molar ratio of the compound with the structure shown in the formula k to lenalidomide is preferably 1: 1; in the present invention, the catalyst used for the substitution reaction is preferably K₂CO₃And KI; the solvent used for the substitution reaction is preferably acetonitrile. In the present invention, the temperature of the substitution reaction is preferably a reflux temperature, and the time is preferably 2 hours.

And carrying out deprotection reaction on the compound with the structure shown as the formula L to obtain the E3 ligand compound containing piperazine ring carbon chain substitution with the structure shown as the formula C-1.

In the present invention, the deprotection reagent used in the deprotection reaction is preferably TFA; the solvent used for the deprotection reaction is preferably DCM. In the present invention, the deprotection reaction is preferably performed at room temperature for 30 min.

In the present invention, the method for preparing the piperazine ring carbon chain-containing heterocyclic compound having the structure represented by the formula C-2 preferably comprises the steps of:

in the present invention, the molar ratio of the compound having the structure represented by formula k to the compound having the structure represented by formula f is preferably 1: 1; in the present invention, the catalyst used for the substitution reaction is preferably K₂CO₃And KI; the solvent used for the substitution reaction is preferably acetonitrile. In the invention, the temperature of the substitution reaction is preferably the reflux temperature, and the time is preferably 8-12 h.

In the present invention, after the substitution reaction, the substitution reaction liquid obtained in the present invention is preferably subjected to a post-treatment, and the post-treatment preferably comprises the steps of:

and (3) sequentially extracting, washing an organic phase, drying and separating by a chromatographic column to obtain a pure compound with a structure shown in a formula m.

In the present invention, the extraction solvent is preferably ethyl acetate and water, and the number of times of extraction is preferably three. In the present invention, the washing detergent is preferably a saturated NaCl solution, and the drying desiccant is preferably anhydrous Na₂SO₄(ii) a In the invention, the stationary phase of the column chromatography separation is preferably 200-300 mesh silica gel, the mobile phase is preferably dichloromethane and methanol, and the volume ratio of the mobile phase is preferably 500-1000: 1.

in the present invention, the deprotection reagent used in the deprotection reaction is preferably TFA, and the solvent used in the deprotection reaction is preferably DCM. In the invention, the deprotection reaction is preferably performed at room temperature, and the time is preferably 0.1-5 h, and more preferably 0.5 h.

In the invention, the preparation method of the piperazine ring carbon chain-containing heterocyclic compound with the structure shown as C-3 comprises the following steps:

in the present invention, the molar ratio of the compound having the structure represented by formula k to trimethylsilyl azide is preferably 3: 1; in the invention, the catalyst of the substitution reaction is preferably KF, and the molar ratio of the KF to the azidotrimethylsilane is preferably 1: 1. In the present invention, the temperature of the substitution reaction is preferably 60 ℃ and the time is preferably 2 hours.

After the substitution reaction, the present invention preferably performs a post-treatment on the obtained substitution reaction solution, and the post-treatment preferably includes the steps of:

and (3) sequentially extracting, washing an organic phase, drying and concentrating the substitution reaction liquid to obtain a pure compound with a structure shown in a formula n.

in the present invention, the catalyst for the hydrogenation reaction is preferably 5 wt% Pd/C; in the present invention, the time for the hydrogenation reaction is preferably 2 hours.

in the present invention, the molar ratio of the compound having the structure represented by formula o to the compound having the structure represented by formula d is preferably 1: 2; the solvent for the substitution reaction is preferably DMF and DIPEA; in the invention, the temperature of the substitution reaction is preferably 90 ℃, and the time is preferably 8-12 h.

And carrying out deprotection reaction on the compound with the structure shown in the formula p to obtain the piperazine ring carbon chain-containing heterocyclic compound with the structure shown in C-3.

In the present invention, the preparation method of the second GPX4 ligand having the structure shown in formula D preferably comprises the following steps:

and (3) carrying out substitution reaction on the compound with the structure shown in the formula t and chloroacetyl chloride to obtain a second GPX4 ligand with the structure shown in the formula D.

In the present invention, the molar ratio of the compound having the structure represented by formula t to chloroacetyl chloride is preferably 0.23: 0.27; the catalyst for the substitution reaction is preferably NaHCO₃(ii) a The temperature of the substitution reaction is preferably room temperature, and the time is preferably 8-12 h.

In the present invention, the method for preparing the compound having the structure represented by formula t preferably comprises the following steps:

carrying out substitution reaction on formyl benzoic acid and m-bromobenzyl bromide to obtain a compound with a structure shown in a formula q;

in the present invention, the molar ratio of p-formylbenzoic acid to m-bromobenzyl bromide is preferably 19.98: 23.98; in the present invention, the catalyst used for the substitution reaction is preferably K₂CO₃(ii) a Said substitutionThe solvent used for the reaction is preferably DMF. In the present invention, the temperature of the substitution reaction is preferably room temperature, and the time is preferably 8 hours.

Under the condition of triethanolamine, carrying out cyclization reaction on a compound with a structure shown as a formula q and a compound with a structure shown as a formula g to obtain a compound with a structure shown as a formula r;

in the present invention, the molar ratio of the triethanolamine, the compound having the structure represented by formula q, and the compound having the structure represented by formula g is preferably 11.61: 9.40: 8.93; in the present invention, the solvent for the cyclization reaction is preferably DCM. In the invention, the temperature of the cyclization reaction is preferably the reflux temperature, and the time is preferably 8-12 h.

A compound having a structure represented by formula r and H₂Carrying out hydrogenation reaction to obtain a compound with a structure shown as a formula t;

in the present invention, the catalyst for the hydrogenation reaction is preferably 5 wt% Pd/C; in the present invention, the time for the hydrogenation reaction is preferably 30 min.

In the invention, the mol ratio of the compound with the structure shown as the formula t to chloroacetyl chloride is preferably 0.1: 10-0.1: 20; the catalyst for the substitution reaction is preferably NaHCO₃(ii) a The temperature of the substitution reaction is preferably room temperature, and the time is preferably 8-12 h.

In the present invention, the preparation method of the third GPX4 ligand having the structure shown in formula E preferably comprises the following steps:

and (3) carrying out substitution reaction on the compound with the structure shown in the formula u and chloroacetyl chloride to obtain a third GPX4 ligand with the structure shown in the formula E.

In the present invention, the molar ratio of the compound having the structure represented by formula u to chloroacetyl chloride is preferably 0.23: 0.27; the catalyst for the substitution reaction is preferably NaHCO₃(ii) a The temperature of the substitution reaction is preferably room temperature, and the time is preferably 8-12 h.

In the present invention, the method for preparing the compound having the structure represented by formula u preferably comprises the following steps:

carrying out cyclization reaction on methylbenzaldehyde and a compound with a structure shown as a formula g to obtain a compound with a structure shown as a formula v;

in the present invention, the molar ratio of the p-tolualdehyde to the compound having the structure represented by the formula g is preferably 11.61: 9.40: 8.93; in the present invention, the solvent for the cyclization reaction is preferably DCM. In the invention, the temperature of the cyclization reaction is preferably the reflux temperature, and the time is preferably 8-12 h.

And carrying out free radical substitution reaction on the compound with the structure shown in the formula v and NBS to obtain the compound with the structure shown in the formula u.

In the present invention, the molar ratio of the compound having the structure represented by formula v to NBS is preferably 1: 1.2; in the present invention, the solvent for the radical substitution reaction is preferably CCl₄. In the invention, the temperature of the free radical substitution reaction is preferably the reflux temperature, and the time is preferably 5-9 h.

In the invention, the preparation method of the piperazine ring carbon chain heterocyclic compound with the structure shown as C-4 comprises the following steps:

in the present invention, the molar ratio of the compound having the structure represented by formula C-2 to 1-Boc-4-bromo-piperidine is preferably 1: 1; in the present invention, the catalyst used for the substitution reaction is preferably K₂CO₃And KI; the solvent used for the substitution reaction is preferably acetonitrile. In the invention, the temperature of the substitution reaction is preferably the reflux temperature, and the time is preferably 8-12 h.

and (3) sequentially extracting, washing an organic phase, drying and separating by a chromatographic column to obtain the pure compound with the structure shown in the formula w.

Carrying out deprotection reaction on the compound with the structure shown in the formula w to obtain an E3 ligand compound containing piperazine ring carbon chain substitution with the structure shown in the formula C-4;

The invention provides application of the GPX4 protein degradation agent in preparation of antitumor drugs. In the invention, the tumor cell is preferably one or more of human fibrosarcoma cell, human breast cancer cell and human lung cancer cell.

The invention provides an anti-tumor cell medicament, which comprises a medicament active component and a medicament auxiliary material; the active pharmaceutical ingredient is the GPX4 protein degradation agent; the invention has no special requirements on the pharmaceutical excipients, and the pharmaceutical excipients well known to the technical personnel in the field can be used. In the invention, the effective content of the pharmaceutical active ingredient in the medicine is 1-50 wt%, preferably 5-30 wt%.

The following examples are provided to illustrate the GPX4 protein degradation agent, its preparation method and application, and an anti-tumor cell drug in detail, but they should not be construed as limiting the scope of the present invention.

In the following examples, the GPX4 degrader intermediate structures referred to are shown in table 1:

TABLE 1GPX4 degradant intermediate structure

The GPX4 degrader structures mentioned are shown in table 2:

TABLE 2GPX4 degradant Structure

Example 1 Synthesis of GPX4 protein degradation agent R1

(1) Synthesis of a first GPX4 ligand

a. Synthesis of Compound represented by formula g

P-formylbenzoic acid (8.00g, 53.29mmol), propiolic acid (4.40g, 79.93mmol) were dissolved in THF (120mL), EDCI (15.32g, 79.93mmol), HOBT (10.80g, 79.93mmol) were added and reacted at room temperature for 14 h. After the reaction is finished, partial solvent is removed by rotary evaporation under reduced pressure, the mixture is extracted by water and ethyl acetate for three times, organic phases are combined, and the mixture is washed by saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. Column separation (P/E. 5/1, 2/1) gave a white solid, which gave the compound of formula g, designated RSL 3-Q1.¹H NMR(400MHz，DMSO-d⁶)δ10.09(s，1H，CHO)，9.17(t，J＝2.68Hz，1H，CONH)，7.80-8.06(m，4H，Ar-H)，4.09(dd，J＝5.52，2.48Hz，2H，CH₂)，3.16(t，J＝2.48Hz，1H，CH)；¹³CNMR(400MHz，DMSO-d⁶)δ193.34，165.61，139.20，138.39，129.91，128.48，81.45，73.54，29.12.

b. Synthesis of Compound of formula i

D-Tryptophan methyl ester hydrochloride (2.57g, 11.75mmol) was dissolved in 15mL DCM, TEA (1.32g, 13.08mmol) was added, and the mixture was stirred at room temperature for 1 hour. Filtration, spin-drying of the filtrate, dissolution in 30mL DCM, addition of RSL3-Q1(2.00g, 10.68mmol), TFA (61mg, 0.53mmol) and

after refluxing for 1 hour, TFA (1.83g, 16.02mmol) was added again and the reaction was refluxed overnight. After the reaction is finished, the reaction product is cooled to room temperature, quenched by 30 percent NaOH solution, separated by organic phase, washed by saturated NaCl solution for three times and anhydrous Na₂SO₄And (5) drying. Column separation (D/M ═ 80/1) gave a white solid, giving the compound of formula i, designated RSL3-Q2(0.50g, 13%).¹H NMR(400MHz，DMSO-d⁶)δ10.65(s，1H，NH)，8.90(t，J＝5.52Hz，1H，CONH)，7.82(d，J＝8.36Hz，2H，Ar-H)，7.46(d，J＝7.68Hz，1H，Ar-H)，7.36(d，J＝8.28Hz，2H，Ar-H)，7.25(d，J＝7.92Hz，1H，Ar-H)，7.02-7.06(m，1H，Ar-H)，6.96-6.70(m，1H，Ar-H)，5.38(s，1H，CH)，4.05(dd，J＝5.52，2.48Hz，2H，CH₂)，3.76-3.81(m，1H，CH)，3.63(s，3H，CH₃)，3.29(s，1H，NH)，3.12(t，J＝2.48Hz，CH)，2.89-3.11(m，2H，CH₂)；¹³CNMR(400MHz，DMSO-d⁶)δ174.25，166.18，146.91，136.61，134.35，133.29，128.67，127.65，126.95，121.40，118.88，118.16，111.55，107.14，81.83，73.26，54.17，52.34，52.13，28.92，25.19.

c. Synthesis of a first GPX4 ligand

RSL3-Q2(0.31g, 0.80mmol) was dissolved with 8mL dry DCM and NaHCO was added₃(74mg, 0.88mmol) and chloroacetyl chloride (0.11g, 0.96mmol) was added portionwise while cooling on ice and reacted at room temperature for 7 h. After the reaction, the reaction solution was quenched with water, extracted with ethyl acetate three times, and the organic phase was washed with saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. Removing part of the solvent under reduced pressure to precipitate a large amount of white solid, filtering, washing the filter cake with a small amount of diethyl ether, and drying to obtain white solid RSL3-Q3(0.24g, 65%), which is the first GPX4 ligand and is recorded as RSL 3-Q3.

(2) Synthesis of Compound represented by the formula A-1

a. Synthesis of intermediate PEG1-4

P-toluenesulfonyl chloride (3.50g, 18.40mmol) was dissolved in 100mL DCM to make solution 1; triethylene glycol dimer (11.00g, 73.00mmol) was dissolved in 100mL DCM and TEA (1.95g, 19.00mmol) and DMAP (46mg, 0.30mmol) were added sequentially to make solution 2, which was equilibrated with stirring for 10min under ice bath. Then, the solution 1 was slowly added to the solution 2 through a dropping funnel in an ice bath, and the dropwise addition was completed within two hours, followed by a reaction at room temperature overnight. After the reaction was complete, quenched with 100mL of water and extracted three times with DCM, the organic phases were combined, washed successively with saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. Column separation (P/E ═ 3/1) gave PEG1-4(2.59g, 46%) as a pale yellow liquid.¹H NMR(400MHz，CDCl₃)δ7.79(d，J＝8.32Hz，2H，Ar-H_a)，7.33(d，J＝7.96Hz，2H，Ar-H_b)，4.15-4.18(m，2H，CH₂)，3.68-3.72(m，4H，CH₂)，3.60(s，4H，CH₂)，3.56-3.58(m，2H，CH₂)，2.44(s，3H，CH₃)，2.07(s，1H，OH)；¹³C NMR(100MHz，CDCl₃)δ144.87，132.95，129.83，127.96，72.47，70.78，70.29，69.15，68.71，61.75，21.64.

b. Synthesis of intermediate PEG1-5

Trimethylazidosilane (2.84g, 24.64mmol) was dissolved in 8mL dry DMF, KF (1.43g, 24.64mmol) was added, mixed well, and stirred at room temperature for 0.5 h. Then, a solution of compound PEG1-4(2.50g, 8.21mmol) in dry DMF (7mL) was added to the reaction and heated to 60 ℃ for 1.5 h. After the reaction is finished, the reaction product is cooled to room temperature, quenched by ice water, extracted by ethyl acetate for three times, the organic phases are combined, washed by 1N NaOH solution and saturated NaCl solution for three times in sequence, and anhydrous Na is used₂SO₄And (5) drying. Column separation (P/E ═ 3/1) gave PEG1-5(0.73g, 51%) as a pale yellow liquid.¹H NMR(400MHz，CDCl₃)δ3.71-3.74(m，2H，CH₂)，3.65-3.68(m，6H，CH₂)，3.59-3.61(m，2H，CH₂)，3.39(t，J＝5.16Hz，2H，CH₂)，2.29(s，1H，OH)；¹³C NMR(100MHz，CDCl₃)δ72.50，70.65，70.37，70.03，61.74，50.65.

c. Synthesis of intermediate PEG1-7

PEG1-5(0.73g, 4.16mmol) was dissolved in 13mL of chloroform, PBr3(3.37g, 12.47mmol) was added dropwise over 5 minutes, and the reaction was refluxed at 50 ℃ overnight. After the reaction is finished, saturated NaHCO is used₃Quenching the solution, extracting with chloroform for three times, mixing the organic phases, washing with saturated NaCl solution and anhydrous Na₂SO₄The solvent mixture, PEG1-6(0.39g), was dried and spun off. Lenalidomide (0.42g, 1.62mmol) in 30mL CH₃CN is dissolved, and the liquid mixture PEG1-6(0.39g, 1.62mmol) and K are added in turn₂CO₃(0.45g, 3.24mmol) and KI (80mg, 0.49mmol) were heated under reflux for 5 h. After the reaction is finished, filtering the mixture,the filtrate is extracted with ethyl acetate and water, the organic phase is washed successively with saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. The column was separated (D/M ═ 50/1) to obtain PEG1-7(0.39g, 23%) as a pale red liquid, which was the compound represented by formula a-1.¹H NMR(400MHz，CDCl₃)δ7.28-7.35(m，2H，Ar-H)，6.87(dd，J＝7.28，1.36Hz，1H，Ar-H)，5.23(dd，J＝13.40，5.12Hz，1H，CH)，4.24(q，J＝44.72，15.52Hz，2H，CH₂)，3.98-4.11(m，2H，CH₂)，3.61-3.70(m，8H，CH₂)，3.38-3.41(m，2H，CH₂)，2.82-3.01(m，2H，CH₂)，2.13-2.37(m，2H，CH₂)；¹³C NMR(100MHz，CDCl₃)δ171.10，170.07，169.78，141.16，132.38，129.48，126.35，118.10，114.40，70.68，69.95，69.92，67.73，52.46，50.71，44.96，39.19，32.09，22.74.

(3) Synthesis of R1

RSL3-Q3(50mg, 0.11mmol), PEG1-7(50mg, 0.12mmol), sodium ascorbate (53mg, 0.26mmol) and copper sulfate pentahydrate (33mg, 0.13mmol) were dissolved in 5mL DMF and reacted at room temperature for 5 h. After the reaction, the reaction solution was quenched with water, extracted three times with ethyl acetate, the organic phases were combined, washed with saturated NaCl solution and anhydrous Na sequentially₂SO₄And (5) drying. Column separation (D/M: 50/1) gave R1(86mg, 89%) as a white solid.¹H NMR(400MHz，DMSO-d⁶)δ11.00(s，1H，NH)，8.96(t，J＝6.20Hz，1H，CONH)，7.77-7.93(m，3H，Ar-H)，7.50-7.64(m，3H，Ar-H)，7.21-7.34(m，2H，Ar-H)，6.96-7.10(m，3H，Ar-H)，6.86(d，J＝7.88Hz，1H，Ar-H)，6.06(s，1H，CH)，5.43(brs，1H，CH)，5.20(dd，J＝13.32，4.92Hz，1H，CH)，4.76(d，J＝14.00Hz，1H，CH_2a)，4.44-4.51(m，5H，CH₂×2&CH_2b)，4.16(q，J＝58.88，17.00Hz，2H，CH₂)，3.74-3.90(m，4H，CH₂×2)，3.41-3.62(m，11H，CH₂×4&CH₃)，2.77-3.01(m，2H，CH_2a)，2.03-2.36(m，2H，CH_2b)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，171.07，169.35，168.19，166.31，160.59，147.48，145.56，143.83，136.87，134.28，133.03，132.70，129.33，127.77，126.26，126.16，123.62，121.93，121.79，119.34，118.48，117.12，111.78，111.14，104.03，69.88，69.81，69.18，67.29，57.03，56.51，53.24，52.54，49.69，46.02，43.76，39.01，35.29，31.87，23.92，22.46；M.P.223-224℃；HRMS(ESI+)：m/z calculated for C₄₄H₄₇ClN₉O₉(M+H)₊：880.3185；found 880.3185.

Example 2-4 Synthesis of protein degradation agent R2-4 of GPX4

Examples 2-4 differ from example 1 in the value of the starting material n in the preparation of a compound having the structure shown by the formula PEG 1-7.

Spectral data of R2:¹H NMR(400MHz，DMSO-d⁶)δ10.93(s，1H，NH)，8.91(t，J＝7.76Hz，1H，CONH)，7.74-7.89(m，3H，Ar-H)，7.46-7.61(m，3H，Ar-H)，7.18-7.30(m，2H，Ar-H)，6.92-7.08(m，3H，Ar-H)，6.81(d，J＝7.88Hz，1H，Ar-H)，6.02(s，1H，CH)，5.39(brs，1H，CH)，5.18(dd，J＝13.24，4.92Hz，1H，CH)，4.72(d，J＝14.00Hz，1H，CH_2a)，4.40-4.48(m，5H，CH₂×2&CH_2b)，4.11(q，J＝57.08，16.76Hz，2H，CH₂)，3.73-3.88(m，4H，CH₂×2)，3.39-3.59(m，15H，CH₂×6&CH₃)，2.74-3.04(m，2H，CH₂)，2.02-2.33(m，2H，CH₂)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，171.07，169.37，168.20，166.26，159.34，145.53，144.04，136.88，134.27，133.07，132.68，129.31，127.75，126.17，126.12，123.61，123.59，121.81，121.78，119.34，118.48，116.98，111.79，110.99，104.00，70.07，70.06，69.95，69.92，69.15，67.23，57.04，56.53，53.22，52.54，49.68，45.99，43.73，39.38，39.03，35.31，25.58，22.48.M.P.199-200℃；HRMS(ESI+)：m/z calculated for C₄₆H₅₁ClN₉O₁₀(M+H)₊：924.3447；found 924.3448.

spectrum of R3Graph data:¹H NMR(400MHz，DMSO-d⁶)δ10.94(s，1H，NH)，8.92(t，J＝5.72Hz，1H，CONH)，7.74-7.89(m，3H，Ar-H)，7.46-7.62(m，3H，Ar-H)，7.18-7.30(m，2H，Ar-H)，6.94-7.06(m，2H，Ar-H)，6.93(d，J＝7.36Hz，1H，Ar-H)，6.81(d，J＝7.88Hz，1H，Ar-H)，6.02(s，1H，CH)，5.42(s，1H，NH)，5.40(brs，1H，CH)，5.18(dd，J＝13.40，5.08Hz，1H，CH)，4.73(d，J＝13.72Hz，1H，CH_2a)，4.40-4.48(m，5H，CH₂×2&CH_2b)，4.16(q，J＝57.76，16.96Hz，2H，CH₂)，3.74-3.89(m，4H，CH₂×2)，3.39-3.59(m，19H，CH₂×8&CH₃)，2.74-3.05(m，2H，CH₂)，2.01-2.33(m，2H，CH₂)；¹³CNMR(400MHz，DMSO-d⁶)δ172.14，171.08，169.36，168.19，166.30，161.36，147.45，145.55，144.09，136.88，134.31，133.04，132.67，129.31，127.76，126.16，126.08，123.60，121.90，121.78，119.34，118.48，116.94，111.77，110.94，104.03，70.18，70.14，70.08，70.05，69.94，69.93，69.14，67.23，57.01，56.52，53.24，52.53，49.68，45.98，43.74，39.02，35.30，31.88，23.92，22.48；M.P.145-146℃；HRMS(ESI+)：m/z calculated for C₄₈H₅₅ClN₉O₁₁(M+H)₊：968.3710；found 968.3717.

spectral data of R4:¹H NMR(400MHz，DMSO-d⁶)δ10.96(s，1H，NH)，8.92(t，J＝5.88Hz，1H，CONH)，7.74-7.90(m，3H，Ar-H)，7.46-7.61(m，3H，Ar-H)，7.18-7.30(m，2H，Ar-H)，6.92-7.04(m，3H，Ar-H)，6.81(d，J＝7.56Hz，1H，Ar-H)，6.03(s，1H，CH)，5.42(s，1H，NH)，5.40(brs，1H，CH)，5.18(dd，J＝13.44，5.12Hz，1H，CH)，4.73(d，J＝14.20Hz，1H，CH_2a)，4.40-4.48(m，5H，CH₂×2&CH_2b)，4.16(q，J＝58.12，16.92Hz，2H，CH₂)，3.75-3.90(m，4H，CH₂×2)，3.41-3.55(m，23H，CH₂×10&CH₃)，2.74-3.06(m，2H，CH₂)，2.02-2.34(m，2H，CH₂)；¹³CNMR(400MHz，DMSO-d⁶)δ172.14，171.08，169.37，168.19，166.31，162.21，149.37，145.57，144.10，136.87，134.27，133.05，132.67，129.30，127.75，126.16，126.09，123.62，121.99，121.78，119.34，118.48，116.94，111.77，110.93，104.64，70.20，70.16，70.15，70.14，70.10，70.08，70.04，69.95，69.15，67.23，57.05，56.52，53.25，52.53，49.69，45.99，43.76，39.02，35.30，31.88，25.40，22.48；M.P.133-134℃；HRMS(ESI+)：m/z calculated for C₅₀H₅₉ClN₉O₁₂(M+H)₊：1012.3972；found 1012.3968.

example 5 Synthesis of protein degradation agent R5 of GPX4

(1) Synthesis of Compound having the Structure represented by the formula A-2

a. Synthesis of intermediate PEG1-1

Triethylene glycol diacetate (3.00g, 19.98mmol) and TEA (6.07mL, 59.94mmol) were dissolved in 15mL DCM and a solution of pTsCl (7.62g, 39.95mmol) in dichloromethane (15mL) was added dropwise over an ice salt bath and the reaction was continued for 4 h. After the reaction was completed, water was added to quench the reaction, DCM was added to extract three times, the organic phases were combined, washed with 1N HCl solution and saturated NaCl solution in order, and then with anhydrous Na₂SO₄And (5) drying. Column separation (P/E-5/1, 2/1) gave PEG1-1(4.72g, 52%) as a white solid, which was subjected to the next reaction without separation.

b. Synthesis of intermediate PEG1-2

Trimethylsilyl azide (1.60g, 13.89mmol) was dissolved in 5mL dry DMF, KF (0.81g, 13.89mmol) was added thereto, and the mixture was stirred at room temperature for 0.5 h. The compound triethylene glycol di-p-toluenesulfonate (1.06g, 2.32mmol) was then added to the system and heated to 60 ℃ for reaction for 2 h. Cooling to room temperature after the reaction is finished, quenching the reaction by ice water, extracting by ethyl acetate for three times, collecting an organic phase, washing by a 1N NaOH solution and a saturated NaCl solution for three times in sequence, and then, using anhydrous Na₂SO₄And (5) drying. Column separation (P/E: 10/1, 6/1) yielded PEG1-2 as a pale green liquid (437.7mg, 94%).¹H NMR(400MHz，CDCl₃)δ3.68-3.70(m，4H，CH₂)，3.39(t，J＝5.08Hz，CH₂)；¹³C NMR(100MHz，CDCl₃)δ70.74，70.13，50.71.

c. Synthesis of intermediate PEG1-3

Compound PEG1-2(1.62g, 8.08mmol) was dissolved in 25mL ethyl acetate, 10mL 1N HCl solution was added, PPh3(2.12g, 8.08mmol) at room temperature, and the reaction was continued for 14 h. After the reaction, the reaction was quenched with water, extracted twice with ethyl acetate, the aqueous phase was adjusted to pH 12-13 with 1N NaOH solution, then extracted three times with dichloromethane, the organic phases were combined, washed with saturated NaCl solution and then with anhydrous Na in sequence₂SO₄Drying, and removing the solvent under reduced pressure to obtain light red liquid PEG1-3(0.79g, 56%), which is not separated for the next reaction.

d. Synthesis of intermediate F-SA-pre

Boc-L-glutamine (3.00g, 12.18mmol) was dissolved in 50mL THF, CDI (2.37g, 14.62mmol) and DMAP (7mg, 0.05mmol) were added sequentially, and the reaction was refluxed for 10 h. After the reaction is finished, the solvent is removed by spinning, the mixture is extracted by ethyl acetate and water for three times, organic phases are combined, and the organic phases are washed by saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column separation (D/M: 50/1) gave F-SA-pre (2.07g, 75%) as a white solid.¹H NMR(400MHz，DMSO-d⁶)δ10.73(s，1H，NH)，7.12(d，J＝8.68Hz，1H，NH)，4.19-4.26(m，1H，CH)，2.50-2.76(m，2H，CH₂)，1.86-1.96(m，2H，CH₂)，1.40(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ173.42，172.96，155.85，78.61，50.86，31.44，28.64，24.89.

e. Synthesis of intermediate F-SA

3-Fluorophthalic anhydride (1.64g, 5.98mmol), F-SA-pre (1.50g, 6.57mmol), and sodium acetate (0.65g, 7.88mmol) were dissolved in 15mL of acetic acid and reacted for 12h under reflux. After the reaction is finished, cooling to room temperature, removing the solvent under reduced pressure, extracting with ethyl acetate and water for three times, combining organic phases, and sequentially using saturated NaHCO₃Saturated solution, NaCl solution washing and anhydrous Na₂SO₄And (5) drying. The solvent was removed under reduced pressure, and ethyl acetate (5mL) and petroleum ether (10mL) were added to the residue, followed by stirring and filtration to give off-white solid F-SA(1.58g，87％)。¹H NMR(400MHz，DMSO-d⁶)δ11.15(s，1H，NH)，7.92-7.97(m，1H，Ar-H)，7.71-7.80(m，2H，Ar-H)，5.14-5.18(m，1H，CH)，2.85-2.94(m，1H，CH_2-a)，2.46-2.63(m，2H，CH_2-b×2)，2.04-2.10(m，1H，CH_2-a)；¹³CNMR(400MHz，DMSO-d⁶)δ173.16，170.12，166.55，164.40，158.56，155.95，138.53，138.45，133.89，123.54，123.35，120.50，120.47，117.55，117.42，49.55，31.36，22.30.

f. Synthesis of intermediate PEG1-8

F-SA (0.44g, 1.59mmol) was dissolved in 8mL dry DMF and PEG1-3(0.33g, 1.91mmol) and DIPEA (0.41g, 3.18mmol) were added sequentially and reacted at 90 ℃ overnight. Cooling to room temperature, adding water, extracting with ethyl acetate for three times, combining the organic phases, washing with saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. Column separation (P/E ═ 3/1) gave PEG1-8(0.23g, 33%) as a yellow oil, a compound having the structure shown in formula a-2.¹H NMR(400MHz，CDCl₃)δ8.46(s，1H，NH)，7.50(t，J＝8.40Hz，1H，Ar-H)，7.11(d，J＝7.12Hz，1H，Ar-H)，6.93(d，J＝8.56Hz，1H，Ar-H)，6.51(t，J＝5.88Hz，1H，NH)，4.91-4.96(m，1H)，3.75(t，J＝5.40Hz，2H，CH₂)，3.68-3.70(m，6H，CH₂×3)，3.48(dd，J＝10.28，5.12Hz，2H，CH₂)，3.38-3.40(m，2H，CH₂)，2.10-2.90(m，4H，CH₂×2)；¹³CNMR(400MHz，CDCl₃)δ171.28，169.29，168.49，167.62，146.83，136.03，132.49，116.79，111.65，110.28，70.72，70.70，70.09，69.58，50.69，48.86，42.38，31.40，22.76.

(2) Synthesis of R5

PEG1-8 and RSL3-Q3 were used as raw materials, and the same procedure and treatment as R1 gave R5 as a yellow solid (47mg, 59.1%).¹H NMR(400MHz，DMSO-d⁶)δ9.27-9.76(m，2H，NH×2)，7.32-7.78(m，6H，Ar-H)，6.98-7.21(m，6H，Ar-H&NH)，6.82(d，J＝8.00Hz，1H，Ar-H)，6.48(brs，1H，NH)，6.03(brs，1H，CH)，5.09(s，1H，CH)，4.82-4.93(m，1H，CH)，4.43-4.58(m，3H，CH_2a&CH₂)，4.01-4.10(m，1H，CH_2b)，3.14-3.79(m，17H，CH₂×7&CH₃)，2.51-2.76(m，3H，CH_2c×2&CH_2d)，1.98-2.13(m，1H，CH_2d)；¹³CNMR(400MHz，DMSO-d⁶)δ172.11，171.89，169.49，169.34，168.65，167.74，160.85，146.72，146.69，136.92，136.13，132.42，128.11，127.46，126.25，126.05，122.25，119.64，118.26，118.21，116.92，116.88，111.67，111.51，111.46，110.12，110.09，70.47，70.35，70.27，69.41，69.35，69.14，69.06，50.36，48.98，42.18，35.26，31.32，29.70，22.73，14.13；M.P.152-153℃；HRMS(ESI+)：m/z calculated for C₄₄H₄₅ClN₉O₁₀(M+H)₊：894.2978；found 894.2978.

Examples 6-8 Synthesis of GPX4 protein degradation Agents R6-8

Examples 6 to 8 are different from example 5 in the value of n as a starting material in the preparation of a compound having a structure represented by the formula PEG 1-8.

Spectral data of R6:¹H NMR(400MHz，DMSO-d⁶)δ9.15-9.43(m，2H，NH×2)，7.77(br，1H，Ar)，7.40-7.63(m，5H，Ar-H)，7.00-7.24(m，6H，Ar-H&NH)，6.86(d，J＝8.60Hz，1H，Ar-H)，6.48(brs，1H，NH)，6.07(brs，1H，CH)，5.17(s，1H，CH)，4.83-4.92(m，1H，CH)，4.61(br，1H，CH_2a)，4.43(br，2H，CH₂)，4.12-4.16(m，1H，CH_2b)，3.79(brs，2H，CH₂)，3.22-3.68(m，19H，CH₂×8&CH₃)，2.60-2.79(m，3H，CH_2c×2&CH_2d)，1.98-2.10(m，1H，CH_2d)；¹³CNMR(400MHz，DMSO-d⁶)δ171.78，171.69，169.34，169.12，168.69，167.72，161.06，146.78，146.75，136.92，136.07，132.41，127.54，126.39，126.08，123.77，123.69，122.27，119.69，119.67，118.31，116.92，116.86，111.61，111.40，110.11，110.08；M.P.112-113℃；HRMS(ESI+)：m/z calculated for C₄₆H₄₉ClN₉O₁₁(M+H)₊：938.3240；found 924.3253.

spectral data of R7:¹H NMR(400MHz，DMSO-d⁶)δ9.10-9.43(m，2H，NH×2)，7.40-7.76(m，6H，Ar-H)，7.01-7.25(m，6H，Ar-H&NH)，6.85(d，J＝8.56Hz，1H，Ar-H)，6.50(brs，1H，NH)，6.07(brs，1H，CH)，5.18(s，1H，CH)，4.82-4.92(m，1H，CH)，4.59-4.64(m，1H，CH_2a)，4.43(brs，2H，CH₂)，4.10-4.13(m，1H，CH_2b)，3.78(br，2H，CH₂)，3.39-3.71(m，23H，CH₂×10&CH₃)，2.59-2.80(m，3H，CH_2c×2&CH_2d)，1.95-2.07(m，1H，CH_2d)；¹³CNMR(400MHz，DMSO-d⁶)δ171.68，171.58，169.36，169.05，167.73，167.70，160.99，146.77，146.74，136.92，136.89，136.10，132.42，132.41，128.29，126.45，126.39，126.05，123.75，122.26，122.22，119.64，118.27，116.87，111.67，111.40，110.17，70.65，70.60，70.46，70.42，70.40，70.36，70.30，70.27，69.28，69.20，69.18，50.20，48.92，42.27，35.18，31.37，31.31，29.67，22.74；M.P.119-120℃；HRMS(ESI+)：m/z calculated for C₄₈H₅₃ClN₉O₁₂(M+H)₊：982.3502；found 982.3503.

spectral data of R8:¹H NMR(400MHz，DMSO-d⁶)δ9.29-9.66(m，2H，NH×2)，7.32-7.88(m，7H，Ar-H)，7.08-7.25(m，5H，Ar-H&NH)，6.89(d，J＝7.96Hz，1H，Ar-H)，6.56(brs，1H，NH)，6.12(brs，1H，CH)，5.20(s，1H，CH)，4.88-5.00(m，1H，CH)，4.50(brs，3H，CH_2a&CH₂)，4.09-4.15(m，1H，CH_2b)，3.84(br，2H，CH₂)，3.38-3.74(m，27H，CH₂×12&CH₃)，2.61-2.84(m，3H，CH_2c×2&CH_2d)，2.01-2.07(m，1H，CH_2d)；¹³CNMR(400MHz，DMSO-d⁶)δ171.64，169.41，169.07，168.57，167.79，167.71，160.16，146.80，146.76，136.98，136.93，136.16，132.47，132.45，126.54，126.48，126.05，122.26，119.58，118.32，118.26，116.95，111.68，111.50，111.41，110.23，110.18，70.64，70.61，70.56，70.49，70.43，70.35，70.32，70.30，70.18，70.17，69.26，69.25，69.18，49.01，42.30，31.43，29.70，29.32，27.22，22.68，14.12；M.P.105-106℃；HRMS(ESI+)：m/z calculated for C₅₀H₅₇ClN₉O₁₃(M+H)₊：1026.3764；found 1026.3783.

example 9 Synthesis of GPX4 protein degradation agent R9

(1) Synthesis of Compound having the Structure represented by the formula A-3

a. Synthesis of intermediate V1

(S) -1- (4-bromobenzene) ethylamine (2.00g, 10.00mmol) was dissolved in EA/H2O (20mL/20mL) and NaHCO was added₃(0.59g, 7.00mmol) and (Boc)2O (2.62g, 12.00mmol), and stirred at room temperature for 1 h. Extracting with ethyl acetate for three times after the reaction is finished, combining organic phases, washing with saturated NaCl solution and anhydrous Na sequentially₂SO₄And (5) drying. Column separation (P/E ═ 40/1) gave V1(1.80g, 60%) as a white solid.¹H NMR(400MHz，CDCl₃)δ7.47(d，J＝8.48Hz，2H，Ar-H_a)，7.20(d，J＝8.36Hz，2H，Ar-H_b)，4.76(br，2H，CH₂)，4.70-4.84(m，2H，CH&NH)，1.37-1.48(m，12H，CH₃&CH₃×3)；¹³C NMR(100MHz，CDCl₃)δ154.97，143.30，131.60，127.59，120.81，79.60，49.80，28.36，22.60.

b. Synthesis of intermediate V2

V1(1.67g, 5.56mmol) was dissolved in 20mL dry DMF and 4-methylthiazole (1.10g, 11.13mmol), Pd (OAc) were added₂(13mg, 0.056mmol) and potassium acetate (1.09g, 11.13mmol), under argon at 90 ℃ for 2 h. After the reaction, cooling to room temperature, adding water, extracting with ethyl acetate for three times, combining organic phases, washing with saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column separation (P/E-15/1) gave V2(0.60g, 47%) as a white solid.¹H NMR(400MHz，CDCl₃)δ8.70(s，1H，Ar-H_a)，7.37-7.43(m，4H，Ar-H×4)，4.84-4.89(m，2H，CH&NH)，2.55(s，3H，CH₃)，1.45-1.50(m，12H，CH₃&CH₃×3)；¹³C NMR(100MHz，CDCl₃)δ155.07，150.22，150.22，148.30，131.76，130.59，129.46，126.22，79.59，49.86，28.39，22.70，16.02.

c. Synthesis of intermediate V4

V2(0.54g, 1.70mmol) was dissolved in 20mL DCM, 10mL TFA was added, and the mixture was stirred at room temperature for 30 min. After the reaction was completed, the solvent was dried to obtain 1.46g of an oily mixture of V3, the mixture of V3 (1.46g, 3.93mmol) was dissolved in 15mL of DMF without separation, and Boc-L-hydroxyproline (0.76g, 3.27mmol), HATU (1.49g, 3.93mmol) and DIPEA (4.23g, 32.70mmol) were added in this order and stirred at room temperature overnight. After the reaction is finished, quenching with water, extracting with ethyl acetate for three times, combining organic phases, and sequentially using 5% citric acid solution and saturated NaHCO₃Washing the solution with saturated NaCl solution, anhydrous Na₂SO₄And (5) drying. The column was separated (D/M. RTM. 60/1) to give V4(0.42g, 58% two steps) as a white solid, which was subjected to the next reaction without separation.

d. Synthesis of intermediate V6

V4(0.41g, 0.95mmol) was dissolved in 20mL DCM, 10mL TFA was added, and the mixture was stirred at room temperature for 30 min. After the reaction was completed, the solvent was dried by spinning to obtain 1.28g of an oily mixture of V5, and without separation, the mixture of V5 (1.46g, 3.93mmol) was dissolved in 15mL of DMF, followed by addition of N-Boc-L-tert-leucine (0.60g, 2.57mmol), HATU (1.17g, 3.08mmol) and DIPEA (3.32g, 25.70mmol), and the mixture was stirred at room temperature overnight. After the reaction is finished, quenching with water, extracting with ethyl acetate for three times, combining organic phases, and sequentially using 5% citric acid solution and saturated NaHCO₃Washing the solution with saturated NaCl solution, anhydrous Na₂SO₄And (5) drying. Column separation (D/M. 60/1) gave V6(0.47g, 90% two steps) as a white solid;¹H NMR(400MHz，CDCl₃)δ8.74(s，1H，Ar-H_a)，7.63(d，J＝7.64Hz，1H，NH)，7.38-7.44(m，4H，Ar-H×4)，5.25(d，J＝9.00Hz，OH)，5.06-5.13(m，1H，CH)，4.79(t，J＝7.80，CH)，4.53(brs，1H，CH)，4.23(d，J＝9.12Hz，1H，CH)，3.58-4.13(m，2H，CH₂)，2.05-2.61(m，2H，CH₂)，2.56(s，3H，CH₃)，1.49(d，J＝6.92Hz，3H，CH₃)，1.43(s，9H，CH₃×3)，1.06(s，9H，CH₃×3)；¹³C NMR(100MHz，CDCl₃)δ172.93，169.52，156.48，150.38，148.14，143.33，131.83，130.64，129.57，126.47，80.50，70.06，58.99，58.19，56.42，48.88，35.19，34.80，28.31，26.46，22.28，15.93.

e. synthesis of intermediate V7

V6(0.45g, 0.83mmol) was dissolved in 20mL DCM, 10mL TFA was added, and the mixture was stirred at room temperature for 30 min. After the reaction was completed, saturated NaHCO was used in ice bath₃The reaction solution was neutralized to weak alkalinity, then extracted with DCM and H2O, followed by washing with saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column separation (D/M: 30/1) gave V7 as an off-white solid (0.35g, 96%).¹H NMR(400MHz，CDCl₃)δ8.69(s，1H，Ar-H_a)，7.76(d，J＝7.48Hz，1H，NH)，7.37-7.42(m，4H，Ar-H×4)，5.05-5.12(m，1H，CH)，4.79(t，J＝7.60，CH)，4.51(brs，1H，CH)，3.60-3.76(m，2H，CH₂)，3.38(s，1H，CH)，2.54(s，3H，CH₃)，2.05-2.49(m，2H，CH₂)，1.50(d，J＝6.92Hz，3H，CH₃)，1.02(s，9H，CH₃×3)；¹³C NMR(100MHz，CDCl₃)δ174.36，170.01，150.24，148.46，143.37，131.60，130.80，129.53，126.39，70.02，60.46，58.58，56.39，48.89，36.06，35.66，26.19，22.32，16.09.

f. Synthesis of intermediate PEG1-10

PEG1-5(1.19g, 6.79mmol) was dissolved in 20mL t-butanol, potassium t-butoxide (0.84g, 7.47mmol) was added, and the mixture was stirred at room temperature for 2 h. Tert-butyl bromoacetate (2.36g, 12.23mmol) was then added and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, diluted with DCM and extracted three times with water, the organic phases were combined, washed sequentially with saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. Column separation (P/E ═ 4/1) gave PEG1-10(0.46g, 24%) as a colorless liquid.¹H NMR(400MHz，CDCl₃)δ4.05(s，2H，CH₂)，3.69-3.75(m，10H，CH₂×5)，3.41(t，J＝5.20Hz，2H，CH₂)，1.50(s，9H，CH₃×3)；¹³CNMR(400MHz，CDCl₃)δ169.65，81.54，70.73，70.68，70.67，70.66，70.03，69.06，50.70，28.11.

g. Synthesis of intermediate PEG1-12

PEG1-10(0.16g, 0.56mmol) was dissolved in 10mL DCM, 1mL TFA was added, and the mixture was stirred at room temperature for 2 h. After the reaction, water was added, DCM was used for extraction three times, the organic phases were combined, washed successively with saturated NaCl solution and anhydrous Na₂SO₄Drying and removal of the solvent gave PEG1-11(74mg, 57%) as a pale yellow liquid mixture, which was subjected to the next reaction without isolation. V7(70mg, 0.16mmol) was dissolved in 5mL DMF, and PEG1-11(74mg, 0.30mmol), HATU (92mg, 0.24mmol) and DIPEA (0.12g, 0.96mmol) were added in this order and reacted at room temperature for 5 h. After the reaction is finished, adding water, extracting with ethyl acetate for three times, combining organic phases, and sequentially using 5% citric acid solution and saturated NaHCO₃Washing the solution with saturated NaCl solution, anhydrous Na₂SO₄And (5) drying. Column separation (D/M ═ 40/1) gave PEG1-12(85mg, 81%) as a colorless liquid, a compound having the structure shown in formula a-3.¹H NMR(400MHz，CDCl₃)δ8.76(s，1H，Ar-H_a)，7.52(d，J＝7.68Hz，1H，NH)，7.38-7.43(m，4H，Ar-H×4)，7.36(d，J＝8.56Hz，1H，NH)，5.06-5.13(m，1H，CH)，4.76(t，J＝7.80，CH)，4.56(d，J＝8.60Hz，1H，CH)，4.53(brs，1H，CH)，4.11(d，J＝11.40Hz，1H，CH_2-a)，4.04(q，J＝15.76，5.16Hz，2H，CH₂)，3.67-3.71(m，10H，CH₂×5)，3.63(dd，J＝11.28，3.72Hz，1H，CH_2-a)，3.39(t，J＝5.16Hz)，2.56(s，3H，CH₃)，2.05-2.54(m，2H，CH_2b)，1.49(d，J＝6.96Hz，3H，CH₃)，1.08(s，9H，CH₃×3)；¹³C NMR(100MHz，CDCl₃)δ171.56，170.55，169.67，150.47，147.92，143.44，131.96，130.49，129.54，126.48，71.16，70.71，70.69，70.53，70.34，70.07，70.04，58.36，57.20，56.60，50.68，48.86，35.40，35.00，26.49，22.26，15.85.

(2) Synthesis of R9

PEG1-12 and RSL3-Q3 were used as raw materials, and the procedure and treatment were performed as in R1 to obtain off-white solid R9(28mg, 25.3%).¹H NMR(400MHz，DMSO-d⁶)δ10.94(s，1H，NH)，8.99(s，1H，Ar-H)，8.93(t，J＝6.28Hz，1H，NH)，8.44(d，J＝7.68Hz，1H，NH)，7.75-7.88(m，3H，Ar-H)，7.36-7.60(m，8H，Ar-H×7&NH)，7.22-7.29(m，1H，Ar)，6.95-7.06(m，2H，Ar-H)，6.03(s，1H，CH)，5.40(brs，1H，CH)，5.14(d，J＝3.52Hz，1H，CH)，4.87-4.94(m，1H，CH)，4.73(d，J＝13.92Hz，1H，CH_2a)，4.55(d，J＝9.56Hz，1H，CH)，4.40-4.48(m，5H，CH₂×2&CH_2b)，4.29(br，1H，CH)，3.93(s，2H，CH₂)，3.79(t，J＝5.00Hz，2H，CH₂)，3.48-3.63(m，15H，CH₂×6&CH₃)，2.46(s，3H，CH₃)，2.04-2.09(m，1H，CH)，1.75-1.81(m，1H，CH)，1.36(d，J＝6.96Hz，3H，CH₃)，0.94(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，170.90，169.47，168.98，168.19，166.31，151.92，148.21，147.47，145.57，145.17，143.02，136.88，134.26，133.04，131.57，130.15，129.28，127.76，126.78，126.17，123.61，121.81，119.34，118.49，111.77，104.02，70.86，70.19，70.04，70.01，69.97，69.23，69.20，59.02，56.99，56.52，56.15，53.25，49.72，48.22，43.76，38.20，36.65，36.22，35.31，26.76，26.68，22.90，16.45；M.P.141-142℃；HRMS(ESI+)：m/z calculated for C₅₆H₆₈ClN₁₀O₁₁S(M+H)₊：1123.4478；found 1123.4496.

Examples 10 to 12 Synthesis of GPX4 protein degradation Agents R10 to 12

Examples 10 to 12 differ from example 9 in the value of the starting material n in the preparation of a compound having a structure represented by the formula PEG 1-12.

Spectral data of R10:¹H NMR(400MHz，DMSO-d⁶)δ10.95(s，1H，NH)，8.99(s，1H，Ar-H)，8.93(t，J＝5.96Hz，1H，NH)，8.45(d，J＝7.60Hz，1H，NH)，7.75-7.91(m，3H，Ar-H)，7.37-7.61(m，8H，Ar-H×7&NH)，7.22-7.33(m，1H，Ar)，6.95-7.09(m，2H，Ar-H)，6.03(s，1H，CH)，5.40(br，1H，CH)，5.14(d，J＝3.32Hz，1H，CH)，4.87-4.94(m，1H，CH)，4.73(d，J＝13.92Hz，1H，CH_2a)，4.55(d，J＝9.48Hz，1H，CH)，4.40-4.49(m，5H，CH₂×2&CH_2b)，4.29(brs，1H，CH)，3.95(s，2H，CH₂)，3.78(t，J＝4.68Hz，2H，CH₂)，3.46-3.63(m，19H，CH₂×8&CH₃)，2.46(s，3H，CH₃)，2.00-2.09(m，1H，CH)，1.75-1.81(m，1H，CH)，1.37(d，J＝7.00Hz，3H，CH₃)，0.94(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.14，170.91，169.47，168.97，168.19，166.30，151.91，148.21，147.46，145.55，145.17，141.66，136.88，134.28，133.06，131.57，130.15，129.28，127.75，126.79，126.17，123.60，121.79，119.34，118.48，111.77，104.03，70.86，70.26，70.20，70.16，70.10，70.01，69.97，69.23，69.18，59.02，56.98，56.50，56.17，53.23，49.71，48.22，43.76，38.20，36.64，36.19，35.31，26.77，26.69，22.91，16.45.M.P.110-111℃；HRMS(ESI+)：m/z calculated for C₅₈H₇₂ClN₁₀O₁₂S(M+H)₊：1167.4740；found 1167.4761.

spectral data of R11:¹H NMR(400MHz，DMSO-d⁶)δ10.93(s，1H，NH)，8.99(s，1H，Ar-H)，8.92(t，J＝4.52Hz，1H，NH)，8.44(d，J＝7.68Hz，1H，NH)，7.74-7.90(m，3H，Ar-H)，7.36-7.60(m，8H，Ar-H×7&NH)，7.21-7.29(m，1H，Ar)，6.94-7.06(m，2H，Ar-H)，6.02(s，1H，CH)，5.40(br，1H，CH)，5.14(br，1H，CH)，4.87-4.94(m，1H，CH)，4.72(d，J＝13.84Hz，1H，CH_2a)，4.55(d，J＝9.56Hz，1H，CH)，4.40-4.48(m，5H，CH₂×2&CH_2b)，4.29(brs，1H，CH)，3.96(s，2H，CH₂)，3.77(t，J＝5.04Hz，2H，CH₂)，3.43-3.61(m，23H，CH₂×10&CH₃)，2.46(s，3H，CH₃)，2.03-2.08(m，1H，CH)，1.74-1.81(m，1H，CH)，1.37(d，J＝7.00Hz，3H，CH₃)，0.94(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，170.91，169.47，168.97，168.19，166.30，151.92，148.21，147.45，145.57，145.17，145.06，136.87，134.27，133.01，131.57，130.14，129.29，127.76，126.78，126.17，123.61，121.76，119.35，118.48，111.78，104.02，70.88，70.74，70.27，70.19，70.17，70.15，70.07，70.04，69.96，69.23，69.17，59.02，56.98，56.51，56.17，53.24，49.69，48.22，43.75，38.20，36.64，36.19，35.30，26.77，26.69，22.91，16.45；M.P.97-98℃；HRMS(ESI+)：m/z calculated for C₆₀H₇₆ClN₁₀O₁₃S(M+H)₊：1211.5003；found 1211.5011.

spectral data of R12:¹H NMR(400MHz，DMSO-d⁶)δ10.93(s，1H，NH)，8.98(s，1H，Ar-H)，8.92(t，J＝5.72Hz，1H，NH)，8.44(d，J＝7.72Hz，1H，NH)，7.74-7.90(m，3H，Ar-H)，7.36-7.61(m，8H，Ar-H×7&NH)，7.21-7.31(m，1H，Ar)，6.94-7.08(m，2H，Ar)，6.02(s，1H，CH)，5.40(brs，1H，CH)，5.14(d，J＝3.48Hz，1H，CH)，4.87-4.94(m，1H，CH)，4.73(d，J＝13.84Hz，1H，CH_2a)，4.55(d，J＝9.56Hz，1H，CH)，4.40-4.48(m，5H，CH₂×2&CH_2b)，4.29(br，1H，CH)，3.96(s，2H，CH₂)，3.77(t，J＝4.96Hz，2H，CH₂)，3.43-3.62(m，27H，CH₂×12&CH₃)，2.46(s，3H，CH₃)，2.02-2.08(m，1H，CH)，1.75-1.81(m，1H，CH)，1.38(d，J＝6.96Hz，3H，CH₃)，0.94(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，170.91，169.47，168.97，168.19，166.28，151.92，148.21，147.47，145.56，145.17，143.12，136.88，134.27，133.09，131.57，130.15，129.29，127.78，126.79，126.17，123.59，121.78，119.35，118.48，111.78，104.03，70.89，70.75，70.30，70.27，70.23，70.21，70.18，70.15，70.07，70.05，69.97，69.24，69.17，59.03，56.98，56.52，56.17，53.23，49.70，48.22，43.76，38.19，36.64，36.19，35.31，26.78，26.70，22.91，16.45.M.P.105-106℃；HRMS(ESI+)：m/z calculated for C₆₂H₈₀ClN₁₀O₁₄S(M+H)₊：1255.5265；found 1255.5278.

example 13 Synthesis of protein degradation agent R13 of GPX4

(1) Synthesis of Compound having the Structure represented by the formula C-1

a. Synthesis of intermediate C7-1

7-bromo-1-heptanol (0.58g, 2.95mmol) was dissolved in 15mL CH3CN, then K was added sequentially₂CO₃(0.74g, 5.36mmol), KI (0.13g, 0.80mmol) and 1-Boc-piperazine (0.50g, 2.68mmol) were heated under reflux for 2 h. After the reaction is finished, cooling to room temperature, filtering, extracting filtrate for three times by using ethyl acetate and water, combining organic phases, washing by using saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column chromatography (D/M-60/1) gave C7-1(505mg, 65%) as a pale yellow waxy solid.¹H NMR(400MHz，CDCl₃)δ3.65(q，J＝11.96，6.20Hz，2H，CH₂)，3.50(t，J＝5.32Hz，4H，CH₂×2)，2.46(br，4H，CH₂×2)，2.40(t，J＝7.52Hz，2H，CH₂)，1.53-1.61(m，4H，CH₂×2)，1.48(s，9H，CH₃×3)，1.32-1.43(m，7H，OH&CH₂×3)；¹³C NMR(100MHz，CDCl₃)δ154.67，79.74，62.92，58.65，58.64，52.93，32.68，29.23，28.42，27.39，26.36，25.64.

b. Synthesis of intermediate C7-2

C7-1(1.10g, 3.66mmol) was dissolved in 20mL DCM, TEA (0.56g, 5.49mmol) was added, and a solution of pTsCl (1.05g, 5.49mmol) in DCM (5mL) was added dropwise with stirring in an ice bath and reacted overnight at room temperature. After the reaction had stopped, it was quenched with water, extracted three times with DCM, the organic phases were combined, washed successively with saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. Column chromatography (D/M-80/1) gave C7-2 as a pale yellow waxy solid (770mg, 46%).¹H NMR(400MHz，CDCl₃)δ7.80(d，J＝8.32Hz，2H，Ar-H×2)，7.36(d，J＝8.00Hz，2H，Ar-H×2)，4.03(t，J＝6.48Hz，2H，CH₂)，3.48(br，4H，CH₂×2)，2.47(s，3H，CH₃)，2.35-2.43(m，6H，CH₂×2，CH₂)，1.54-1.69(m，2H，CH₂)，1.45-1.54(m，11H，CH₃×3，CH₂)，1.27-1.36(m，6H，CH₂×3)；¹³C NMR(100MHz，CDCl₃)δ154.67，144.63，133.22，129.79，127.86，79.72，70.57，58.55，58.55，52.93，28.78，28.76，28.42，27.19，36.36，25.27，21.64.

c. Synthesis of intermediate C7-3

Lenalidomide (0.18g, 0.68mmol) was dissolved in 10mL CH3CN, and then C7-2(0.31g, 0.68mmol), K were added sequentially₂CO₃(0.11g, 0.82mmol) and KI (34mg, 0.20mmol) were reacted for 2h under reflux. After the reaction is finished, cooling to room temperature, filtering, extracting filtrate for three times by using ethyl acetate and water, combining organic phases, washing by using saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column chromatography (D/M ═ 60/1) gave C7-3(259mg, 70%) as a white solid.¹H NMR(400MHz，CDCl₃)δ7.29-7.36(m，2H，Ar-H×2)，6.87(dd，J＝7.40，1.28Hz，1H，Ar-H)，5.20(dd，J＝13.44，5.16Hz，1H，CH)，4.26(q，J＝52.32，15.40Hz，2H，CH₂)，3.73-3.83(m，4H，CH₂×2)，3.48(t，J＝4.52Hz，4H，CH₂×2)，2.96-3.02(m，1H，CH_2-a)，2.81-2.91(m，1H，CH_2-b)，2.35-2.45(m，6H，CH₂×2，CH₂)，2.25-2.32(m，1H，CH_2-b)，2.15-2.21(m，1H，CH_2-a)，1.51-1.54(m，2H，CH₂)，1.47(s，9H，CH₃×3)，1.29-1.35(m，6H，CH₂×3)；¹³C NMR(100MHz，CDCl₃)δ170.98，169.91，169.81，154.64，141.17，132.40，129.49，126.28，118.09，114.47，79.74，58.73，58.57，52.90，52.49，45.02，40.56，32.23，29.47，28.93，28.41，27.82，27.23，26.74，22.90.

d. Synthesis of intermediate C7-4

C7-3(0.20g, 0.37mmol) was dissolved in 10mL DCM, 1mL TFA was added, and the mixture was stirred at room temperature for 30 min. After the completion of the reaction, the solvent was removed under reduced pressure to obtain a pale yellow liquid mixture C7-4, which was subjected to the next reaction without separation.

e. Synthesis of intermediate RSL3-S1

4-formylbenzoic acid (3.00g, 19.98mmol) and anhydrous K₂CO₃(4.14g, 29.97mmol) was dissolved in 20mL DMF and m-bromobenzyl bromide (5.99g, 23.98mmol) was added and reacted at room temperature for 8 h. After the reaction, water was added, extraction was carried out three times with ethyl acetate, and the organic phases were combined, washed successively with saturated NaCl solution and anhydrous Na₂SO₄Drying, removing solvent to obtain white waxy solid RSL3-S1(6.08g, 95.3%), and performing the next reaction without separation;

f. synthesis of intermediate RSL3-S2

Adding tryptophan methyl ester hydrochloride (2.28g, 8.93mmol) into 30mL DCM, adding TEA (1.18g, 11.61mmol), stirring at room temperature for 1h, filtering, drying the filtrate by spinning, dissolving with 40mL DCM, and sequentially adding RSL3-S1(3.00g, 9.40mmol),

molecular sieves and TFA (0.11g, 0.94mmol) were refluxed for 1h and TFA (3.22g, 28.20mmol) was added again and the reaction was continued at reflux overnight. After the reaction is finished, cooling to room temperature, quenching the reaction by using 30% NaOH solution, extracting and separating an organic phase, and washing by using saturated NaCl solution and anhydrous Na in sequence₂SO₄Drying and column separation (P/E. 10/1) gave a pale yellow waxy solid, RSL3-S2(2.5g, 54%).¹H NMR(400MHz，CDCl₃)δ8.03(d，J＝8.24Hz，2H，Ar-H)，7.70(s，1H，Ar-H)，7.57-7.60(m，2H，Ar-H&NH)，7.49(d，J＝7.92Hz，1H，Ar-H)，7.36-7.40(m，3H，Ar-H)，7.25-7.29(m，2H，Ar-H)，7.14-7.21(m，2H，Ar-H)，5.48(s，1H，CH)，5.33(s，2H，CH₂)，3.97(t，J＝6.12，1H，CH)，3.74(s，3H，CH₃)，3.14-3.33(m，2H，CH₂)；¹³C NMR(100MHz，CDCl₃)δ174.00，165.89，147.39，138.24，136.26，132.36，131.38，131.02，130.19，130.18，129.59，128.50，126.91，126.61，122.65，122.22，119.68，118.35，110.97，108.60，65.74，54.63，52.66，52.17，24.57.

g. Synthesis of intermediate RSL3-S3

RSL3-S2(0.50g, 0.96mmol) was dissolved in 15mL MeOH, 100mg of 5% Pd/C was added, and the mixture was hydrogenated for 30 min. After completion of the reaction, the reaction mixture was filtered, and the filtrate was subjected to column separation (D/M: 30/1) to obtain RSL3-S3(0.15g, 45%) as a white solid.¹H NMR(400MHz，DMSO-d⁶)δ10.65(s，1H，COOH)，7.90(d，J＝8.28Hz，2H，Ar-H)，7.46(d，J＝7.64Hz，1H，Ar-H)，7.40(d，J＝8.24Hz，1H，Ar-H)，7.25(d，J＝7.92Hz，2H，Ar-H)，6.96-7.06(m，2H，Ar-H)，5.40(s，1H，CH)，3.79(dd，J＝7.16，5.32Hz，1H，CH)，3.63(s，3H，CH₃)，2.89-3.11(m，2H，CH₂)；¹³CNMR(400MHz，DMSO-d⁶)δ174.26，167.68，148.40，136.61，134.16，130.23，129.72，128.91，126.91，121.44，118.92，118.17，111.56，107.13，54.21，52.38，52.15，25.13.

h. Synthesis of intermediate RSL3-S4

RSL3-S3(80mg, 0.23mmol) was dissolved with 5mL DCM and NaHCO was added₃(23mg, 0.27mmol), chloroacetyl chloride (31mg, 0.27mmol) was added portionwise in ice bath, then turned to room temperature for reaction overnight. After the reaction is finished, the organic phase is extracted and separated, and is washed by saturated NaCl solution and anhydrous Na in sequence₂SO₄The column was dried and isolated (D/M. 30/1) to give RSL3-S4 as an off-white solid (70mg, 71%).¹H NMR(400MHz，DMSO-d⁶)δ12.84(s，1H，COOH)，10.93(s，1H，NH)，7.82(d，J＝8.56Hz，2H，Ar-H)，7.47-7.54(m，3H，Ar-H)，7.23(d，J＝7.72Hz，1H，Ar-H)，6.95-7.05(m，2H，Ar-H)，6.04(s，1H，CH)，5.40(s，1H，CH)，4.73(d，J＝13.80Hz，1H，CH_2-a)，4.42(d，J＝14.08Hz，1H，CH_2-b)，3.52-3.59(m，5H，CH₂&CH₃)；¹³CNMR(400MHz，DMSO-d⁶)δ168.26，167.46，162.73，149.14，136.91，134.05，129.82，129.61，126.47，126.17，121.85，119.37，118.52，111.82，104.15，57.06，56.65，53.25，43.74，23.92.

(2) Synthesis of R13

RSL3-S4(77mg, 0.18mmol) was dissolved in 8mL DMF and C7-4(80mg, 0.18mmol), HATU (96mg, 0.25mmol) and DIPEA (140mg, 1.08mmol) were added sequentially and stirred at room temperature for 2 h. After the reaction, the reaction solution was extracted three times with water and ethyl acetate, and the organic phases were combined, washed with saturated NaCl solution and anhydrous Na sequentially₂SO₄And (5) drying. Column chromatography (D/M-50/1) gave R13(83mg, 54%) as a white solid.¹H NMR(400MHz，DMSO-d⁶)δ10.97(s，1H，NH)，7.47-7.59(m，3H，Ar-H)，7.18-7.41(m，4H，Ar-H)，6.96-7.09(m，2H，Ar-H)，6.92(d，J＝7.16Hz，1H，Ar-H)，6.81(d，J＝7.72Hz，1H，Ar-H)，6.03(brs，1H，CH)，5.43(s，1H，NH)，5.40(br，1H，CH)，5.17(dd，J＝13.32，5.00Hz，1H，CH)，4.73(d，J＝13.64Hz，1H，CH_2a)，4.42(d，J＝13.93Hz，1H，CH_2b)，4.15(dd，J＝54.80，16.88Hz，2H，CH₂)，3.51-3.68(m，7H，CH₂×2&CH₃)，3.36-3.41(m，2H，CH₂)，3.19-3.32(m，2H，CH₂)，2.96-3.06(m，1H，CH_2c)，2.73-2.79(m，1H，CH_2d)，2.18-2.43(m，7H，CH₂×3&CH_2d)，2.01-2.08(m，1H，CH_2c)，1.18-1.45(m，10H，CH₂×5)；¹³CNMR(400MHz，DMSO-d⁶)δ172.14，171.03，169.38，168.22，166.92，162.46，144.09，136.82，134.45，132.71，129.31，128.03，127.53，126.28，126.21，126.06，121.78，119.38，118.50，116.91，111.87，110.90，107.32，104.03，67.34，57.97，57.09，56.48，55.38，53.25，52.58，49.07，46.09，43.83，31.93，28.94，27.82，27.06，26.71，26.47，23.94，22.54；M.P.254-255℃；HRMS(ESI+)：m/z calculated for C₄₆H₅₃ClN₇O₇(M+H)₊：850.3695；found 850.3708.

Examples 14-18 Synthesis of protein degradation Agents R14-18 of GPX4

Examples 14 to 18 are different from example 13 in the value of n as a starting material in the preparation of a compound having a structure represented by the formula C-1.

Spectral data of R14:¹H NMR(400MHz，DMSO-d6)δ10.96(s，1H，NH)，7.46-7.59(m，3H，Ar-H)，7.18-7.41(m，4H，Ar-H)，6.96-7.10(m，2H，Ar-H)，6.92(d，J＝7.20Hz，1H，Ar-H)，6.81(d，J＝7.80Hz，1H，Ar-H)，6.03(br，1H，CH)，5.42(s，1H，NH)，5.40(br，1H，CH)，5.17(dd，J＝13.36，5.04Hz，1H，CH)，4.73(d，J＝13.76Hz，1H，CH2a)，4.42(d，J＝14.12Hz，1H，CH2b)，4.15(dd，J＝54.12，16.92Hz，2H，CH2)，3.51-3.68(m，7H，CH2×2&CH3)，3.35-3.42(m，2H，CH2)，3.18-3.31(m，2H，CH2)，2.97-3.06(m，1H，CH2c)，2.74-2.79(m，1H，CH2d)，2.21-2.42(m，7H，CH2×3&CH2d)，2.01-2.08(m，1H，CH2c)，1.19-1.45(m，14H，CH2×7)；13CNMR(400MHz，DMSO-d6)δ171.77，170.65，169.00，167.85，162.40，143.72，136.45，134.08，132.34，128.94，127.66，127.16，125.91，125.84，125.69，121.41，119.01，118.12，116.54，111.50，110.54，106.95，103.66，65.78，57.53，56.72，56.11，54.25，52.88，52.21，48.69，45.71，43.45，31.56，28.90，28.90，28.71，28.71，27.49，26.81，26.39，23.56，22.18.M.P.242-243℃；HRMS(ESI+)：m/z calculated for C48H57ClN7O7(M+H)+：878.4008；found 878.4022.

spectral data of R15:¹H NMR(400MHz，DMSO-d⁶)δ10.96(s，1H，NH)，7.46-7.58(m，3H，Ar-H)，7.18-7.41(m，4H，Ar-H)，6.95-7.08(m，2H，Ar-H)，6.92(d，J＝7.40Hz，1H，Ar-H)，6.81(d，J＝7.92Hz，1H，Ar-H)，6.03(br，1H，CH)，5.43(s，1H，NH)，5.40(br，1H，CH)，5.17(dd，J＝13.36，5.04Hz，1H，CH)，4.73(d，J＝13.84Hz，1H，CH_2a)，4.42(d，J＝13.92Hz，1H，CH_2b)，4.15(dd，J＝53.52，16.92Hz，2H，CH₂)，3.51-3.68(m，7H，CH₂×2&CH₃)，3.36-3.40(m，2H，CH₂)，3.17-3.31(m，2H，CH₂)，2.97-3.06(m，1H，CH_2c)，2.73-2.78(m，1H，CH_2d)，2.19-2.40(m，7H，CH₂×3&CH_2d)，2.01-2.08(m，1H，CH_2c)，1.18-1.45(m，18H，CH₂×9)；¹³CNMR(400MHz，DMSO-d⁶)δ172.11，171.01，169.36，169.06，168.21，161.58，144.09，136.84，134.46，132.71，129.29，127.97，127.47，127.32，126.22，126.07，121.79，119.37，118.49，116.91，111.86，110.91，105.89，104.02，74.29，58.13，57.08，56.50，54.64，53.23，52.58，47.57，46.06，43.80，31.94，29.45，29.42，29.40，29.34，29.13，27.88，27.34，26.79，26.60，23.93，22.56；M.P.149-150℃；HRMS(ESI+)：m/z calculated for C₅₀H₆₁ClN₇O₇(M+H)₊：906.4321；found 906.4327.

spectral data of R16:¹H NMR(400MHz，DMSO-d⁶)δ10.97(s，1H，NH)，7.46-7.61(m，3H，Ar-H)，7.18-7.41(m，4H，Ar-H)，6.96-7.11(m，2H，Ar-H)，6.93(d，J＝7.40Hz，1H，Ar-H)，6.81(d，J＝7.84Hz，1H，Ar-H)，6.03(brs，1H，CH)，5.43(s，1H，NH)，5.41(br，1H，CH)，5.17(dd，J＝13.36，5.04Hz，1H，CH)，4.74(d，J＝13.84Hz，1H，CH_2a)，4.42(d，J＝14.12Hz，1H，CH_2b)，4.15(dd，J＝53.12，16.92Hz，2H，CH₂)，3.51-3.68(m，7H，CH₂×2&CH₃)，3.35-3.44(m，2H，CH₂)，3.19-3.31(m，2H，CH₂)，2.97-3.06(m，1H，CH_2c)，2.73-2.80(m，1H，CH_2d)，2.21-2.41(m，7H，CH₂×3&CH_2d)，2.00-2.08(m，1H，CH_2c)，1.17-1.45(m，20H，CH₂×10)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，171.03，169.37，168.23，162.46，144.11，136.82，134.46，132.72，129.31，128.03，127.53，126.29，126.22，126.07，121.78，119.38，118.50，116.91，111.87，110.91，107.33，104.04，67.35，57.97，57.09，56.48，54.68，53.25，52.58，49.07，46.08，43.83，31.94，29.45，29.44，29.43，29.42，29.36，29.15，27.88，27.24，26.80，25.98，23.94，22.56.M.P.132-133℃；HRMS(ESI+)：m/z calculated for C₅₁H₆₃ClN₇O₇(M+H)₊：920.4478；found 920.4495.

spectral data of R17:¹H NMR(400MHz，DMSO-d⁶)δ10.95(s，1H，NH)，7.47-7.57(m，3H，Ar-H)，7.17-7.38(m，4H，Ar-H)，6.95-7.06(m，2H，Ar-H)，6.92(d，J＝7.16Hz，1H，Ar-H)，6.80(d，J＝7.88Hz，1H，Ar-H)，6.03(br，1H，CH)，5.41(s，1H，NH)，5.39(brs，1H，CH)，5.16(dd，J＝13.36，5.08Hz，1H，CH)，4.72(d，J＝13.88Hz，1H，CH_2a)，4.42(d，J＝14.40Hz，1H，CH_2b)，4.16(dd，J＝52.96，16.92Hz，2H，CH₂)，3.51-3.68(m，7H，CH₂×2&CH₃)，3.33-3.40(m，2H，CH₂)，3.17-3.30(m，2H，CH₂)，2.96-3.06(m，1H，CH_2c)，2.73-2.78(m，1H，CH_2d)，2.21-2.39(m，7H，CH₂×3&CH_2d)，2.00-2.08(m，1H，CH_2c)，1.35-1.46(m，4H，CH₂×2)，1.21-1.24(m，18H，CH₂×9)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，171.03，169.36，169.08，168.22，144.11，136.83，134.47，132.72，129.30，127.99，127.48，126.29，126.22，126.07，121.78，119.37，118.50，116.90，111.87，110.90，105.82，104.03，61.18，58.17，57.08，56.49，54.65，53.25，52.57，47.69，46.06，43.82，31.94，29.84，29.46，29.45，29.43，29.42，29.37，29.15，27.89，27.35，26.81，26.63，23.94，22.57.M.P.123-124℃；HRMS(ESI+)：m/z calculated for C₅₂H₆₅C1N₇O₇(M+H)₊：934.4634；found 934.4650.

spectral data of R18:¹H NMR(400MHz，DMSO-d⁶)δ10.96(s，1H，NH)，7.47-7.61(m，3H，Ar-H)，7.18-7.40(m，4H，Ar-H)，6.97-7.07(m，2H，Ar-H)，6.92(d，J＝4.88Hz，1H，Ar-H)，6.80(d，J＝5.28Hz，1H，Ar-H)，6.03(br，1H，CH)，5.42(s，1H，NH)，5.40(br，1H，CH)，5.16(dd，J＝8.92，3.36Hz，1H，CH)，4.73(d，J＝9.36Hz，1H，CH_2a)，4.42(d，J＝9.32Hz，1H，CH_2b)，4.15(dd，J＝52.04，11.20Hz，2H，CH₂)，3.51-3.66(m，7H，CH₂×2&CH₃)，3.35-3.39(m，2H，CH₂)，3.21-3.30(m，2H，CH₂)，2.98-3.04(m，1H，CH_2c)，2.74-2.78(m，1H，CH_2d)，2.21-2.39(m，7H，CH₂×3&CH_2d)，2.02-2.07(m，1H，CH_2c)，1.36-1.44(m，4H，CH₂×2)，1.19-1.26(m，20H，CH₂×10)；¹³CNMR(400MHz，DMSO-d⁶)δ172.13，171.02，169.36，168.22，166.91，163.63，144.10，139.42，136.82，134.95，132.71，129.85，129.30，127.49，126.21，126.06，121.81，119.37，118.49，116.90，111.86，110.90，107.33，104.03，77.52，58.10，57.08，56.48，54.65，53.24，52.57，49.06，46.06，43.82，31.93，29.55，29.49，29.48，29.47，29.47，29.47，29.38，29.36，29.14，27.88，27.31，26.80，26.60，23.94，22.56；M.P.117-118℃；HRMS(ESI+)：m/z calculated for C₅₃H₆₇ClN₇O₇(M+H)₊：948.4791；found 948.4811.

example 19 Synthesis of protein degradation agent R19 of GPX4

(1) Synthesizing a compound with a structure shown as a formula C-3;

a. synthesis of intermediate C11-2-1

Trimethylsilyl azide (0.92g, 7.98mmol) was dissolved in 15mL dry DMF, KF (0.47g, 7.98mmol) was added, and the mixture was stirred at room temperature for 30min, followed by addition of C11-2(1.36g, 2.66mmol) and reaction at 60 ℃ for 2 h. After the reaction is finished, quenching the reaction by using a cold NaOH solution, extracting by using ethyl acetate for three times, combining organic phases, washing by using a saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Vacuum concentrating to obtain light yellow liquid C11-2-1(0.90g, 89%), and performing the next reaction without separation;

b. synthesis of intermediate C11-2-2

C11-2-1(0.90g, 2.36mmol) was dissolved in 15mL of methanol, 5% Pd/C (180mg) was added, and hydrogenation was carried out for 2 h. After the reaction is finished, filtering to remove palladium carbon, concentrating the filtrate to obtain 0.73g of gray solid, and carrying out the next reaction without separation;

c. synthesis of intermediate C11-5

F-SA (0.20g, 0.72mmol) was dissolved in 5mL dry DMF and C11-2-2(0.51g, 1.44mmol) and DIPEA (0.19g, 1.44mmol) were added sequentially and reacted at 90 ℃ overnight. After the reaction is finished, quenching the reaction by water, extracting by ethyl acetate for three times, combining organic phases, washing by saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column chromatography (D/M ═ 60/1) gave C11-5(110mg, 25%) as a yellow solid.¹H NMR(400MHz，CDCl₃)δ8.70(s，1H，NH)，7.48-7.52(m，1H，Ar-H)，7.10(d，J＝7.04Hz，1H，Ar-H)，6.89(d，J＝8.52Hz，1H，Ar-H)，6.24(t，J＝5.40Hz，NH)，4.91-4.95(m，1H，CH)，3.49(br，4H，CH₂×2)，3.27(q，J＝12.64，6.84Hz，2H，CH₂)，2.70-2.92(m，3H，CH_2-a×2&CH_2-b)，2.35-2.51(m，6H，CH₂×2&CH₂)，2.11-2.17(m，1H，CH_2-b)，1.63-1.71(m，2H，CH₂)，1.50-1.58(m，2H，CH₂)，1.47(s，9H，CH₃×3)，1.30-1.45(m，14H，CH₂×7)；¹³C NMR(100MHz，CDCl₃)δ171.13，169.50，168.46，167.64，154.69，147.03，136.07，132.49，116.62，111.32，109.83，79.73，58.63，52.85，52.85，48.89，42.61，31.48，29.47，29.44，29.36，29.17，29.14，29.14，28.42，27.46，26.81，26.45，22.84.

d. Synthesis of intermediate C11-6

C11-5(0.10g, 0.16mmol) was dissolved in 10mL DCM, 1mL TFA was added, and the mixture was stirred at room temperature for 30 min. After the reaction is finished, removing the solvent under reduced pressure to obtain a light yellow liquid mixture C11-6, and carrying out the next reaction without separation;

(2) synthesis of R19

The same procedure and work-up as R13 gave R19 as a yellow solid (68%) starting from C11-6 and RSL 3-S4.¹H NMR(400MHz，DMSO-d⁶)δ11.11(s，1H，NH)，10.97(s，1H，NH)，7.57-7.59(m，2H，Ar-H)，7.49(d，J＝5.20Hz，2H，Ar-H)，7.24-7.41(m，3H，Ar-H)，7.09(d，J＝5.76Hz，1H，Ar-H)，7.02-7.07(m，2H，Ar-H)，6.96-6.99(m，1H，Ar-H)，6.52(t，J＝3.88Hz，1H，NH)，6.04(brs，1H，CH)，5.40(br，1H，CH)，5.06(dd，J＝8.56，3.64Hz，1H，CH)，4.74(d，J＝9.36Hz，1H，CH_2a)，4.43(d，J＝9.28Hz，1H，CH_2b)，3.52-3.59(m，6H，CH₂×3)，3.27-3.37(m，5H，CH₂&CH₃)，2.86-2.92(m，1H，CH_2c)，2.49-2.61(m，2H，CH_2d×2)，2.25-2.42(m，6H，CH₂×3)，2.01-2.05(m，1H，CH_2c)，1.54-1.59(m，2H，CH₂)，1.39(br，2H，CH₂)，1.24-1.35(m，14H，CH₂×7)；¹³CNMR(400MHz，DMSO-d⁶)δ173.26，172.15，170.54，169.41，169.10，168.22，167.76，146.89，136.82，136.73，134.46，132.65，128.03，127.50，127.30，126.29，126.22，121.78，119.37，118.50，117.64，111.87，110.84，109.46，104.04，58.12，57.09，56.48，54.64，53.25，49.00，43.83，43.45，42.29，40.53，31.45，29.43，29.41，29.20，29.12，27.33，26.76，26.63，23.94，22.63，21.97；M.P.135-136℃；HRMS(ESI+)：m/z calculated for C₅₀H₅₉ClN₇O₈(M+H)₊：920.4114；found 920.4113.

Examples 20 to 22 Synthesis of GPX4 protein degradation Agents R20 to 22

Examples 20 to 22 are different from example 19 in the value of n as a starting material in the production of a compound having a structure represented by the formula C-3.

Spectral data of R20:¹H NMR(400MHz，DMSO-d⁶)δ11.11(s，1H，NH)，10.98(s，1H，NH)，7.57-7.59(m，2H，Ar-H)，7.49(d，J＝5.16Hz，2H，Ar-H)，7.24-7.42(m，3H，Ar-H)，7.09(d，J＝5.76Hz，1H，Ar-H)，7.02-7.07(m，2H，Ar-H)，6.96-6.99(m，1H，Ar-H)，6.52(t，J＝3.84Hz，1H，NH)，6.04(br，1H，CH)，5.41(brs，1H，CH)，5.06(dd，J＝8.60，3.64Hz，1H，CH)，4.74(d，J＝9.36Hz，1H，CH_2a)，4.43(d，J＝9.28Hz，1H，CH_2b)，3.52-3.59(m，6H，CH₂×3)，3.27-3.38(m，5H，CH₂&CH₃)，2.86-2.92(m，1H，CH_2c)，2.49-2.61(m，2H，CH_2d×2)，2.11-2.44(m，6H，CH₂×3)，2.01-2.05(m，1H，CH_2c)，1.54-1.59(m，2H，CH₂)，1.40(br，2H，CH₂)，1.24-1.35(m，16H，CH₂×8)；¹³CNMR(400MHz，DMSO-d⁶)δ173.26，172.15，170.54，169.42，169.12，168.22，167.76，146.89，136.82，136.73，134.46，132.65，128.03，127.53，127.30，126.29，126.22，121.78，119.37，118.50，117.63，111.87，110.84，109.47，104.04，58.12，57.09，56.48，54.68，53.25，49.01，43.83，43.15，42.29，40.53，31.46，29.45，29.43，29.37，29.21，29.12，27.26，26.76，26.63，23.94，22.63，21.97；M.P.128-129℃；HRMS(ESI+)：m/z calculated for C₅₁H₆₁ClN₇O₈(M+H)₊：934.4270；found 934.4282.

spectral data of R21:¹H NMR(400MHz，DMSO-d⁶)δ11.10(s，1H，NH)，10.96(s，1H，NH)，7.57-7.61(m，2H，Ar-H)，7.47-7.49(m，2H，Ar-H)，7.24-7.40(m，3H，Ar-H)，7.09(d，J＝5.72Hz，1H，Ar-H)，7.02-7.05(m，2H，Ar-H)，6.96-6.99(m，1H，Ar-H)，6.53(t，J＝3.88Hz，1H，NH)，6.03(brs，1H，CH)，5.40(brs，1H，CH)，5.06(dd，J＝8.60，3.68Hz，1H，CH)，4.73(d，J＝9.36Hz，1H，CH_2a)，4.42(d，J＝9.32Hz，1H，CH_2b)，3.51-3.59(m，6H，CH₂×3)，3.33(s，3H，CH₃)，3.28(dd，J＝8.84，4.40Hz，2H，CH₂)，2.86-2.92(m，1H，CH_2c)，2.54-2.61(m，2H，CH_2d×2)，2.25-2.40(m，6H，CH₂×3)，2.01-2.05(m，1H，CH_2c)，1.54-1.59(m，2H，CH₂)，1.35-1.38(m，2H，CH₂)，1.23-1.29(m，18H，CH₂×9)；¹³CNMR(400MHz，DMSO-d⁶)δ173.26，172.15，170.54，169.41，168.22，167.76，167.43，146.89，136.83，136.72，132.65，132.18，132.03，129.15，127.48，126.29，126.22，121.77，119.37，118.49，117.63，111.86，110.83，109.47，104.03，58.14，57.08，56.49，54.66，53.25，49.00，43.83，43.12，42.29，40.53，31.45，30.27，29.47，29.44，29.20，29.12，28.84，27.68，27.33，26.76，23.71，22.87，22.63；M.P.84-85℃；HRMS(ESI+)：m/z calculated for C₅₂H₆₃ClN₇O₈(M+H)₊：948.4427；found 948.4449.

spectral data of R22:¹H NMR(400MHz，DMSO-d⁶)δ11.10(s，1H，NH)，10.98(s，1H，NH)，7.56-7.58(m，2H，Ar-H)，7.48(d，J＝5.20Hz，2H，Ar-H)，7.24-7.40(m，3H，Ar-H)，7.08(d，J＝5.72Hz，1H，Ar-H)，7.01-7.06(m，2H，Ar-H)，6.96-6.98(m，1H，Ar-H)，6.52(t，J＝3.84Hz，1H，NH)，6.04(brs，1H，CH)，5.40(brs，1H，CH)，5.05(dd，J＝8.60，3.68Hz，1H，CH)，4.73(d，J＝9.32Hz，1H，CH_2a)，4.42(d，J＝9.28Hz，1H，CH_2b)，3.49-3.59(m，6H，CH₂×3)，3.24-3.33(m，5H，CH₂&CH₃)，2.86-2.92(m，1H，CH_2c)，2.52-2.61(m，2H，CH_2d×2)，2.15-2.45(m，6H，CH₂×3)，2.00-2.04(m，1H，CH_2c)，1.53-1.58(m，2H，CH₂)，1.39(br，2H，CH₂)，1.22-1.34(m，20H，CH₂×10)；¹³CNMR(400MHz，DMSO-d⁶)δ173.26，172.15，170.53，169.41，169.10，168.22，167.76，146.88，136.82，136.72，134.46，132.64，128.01，127.50，127.29，126.28，126.21，121.81，119.37，118.48，117.62，111.86，110.83，109.46，104.02，58.07，57.09，56.48，54.66，53.24，49.00，43.82，43.14，42.29，40.51，31.45，29.48，29.47，29.44，29.38，29.27，29.20，29.12，27.28，26.76，26.47，23.93，22.63，21.59；M.P.116-117℃；HRMS(ESI+)：m/z calculated for C₅₃H₆₅ClN₇O₈(M+H)₊：962.4583；found 962.4600.

example 23 Synthesis of protein degradation agent R23 of GPX4

(1) Synthesizing a compound with a structure shown as a formula C-2;

a. synthesis of intermediate C7-7

V7(0.21g, 0.47mmol) was dissolved in 10mL CH3CN, and then C7-2(0.22g, 0.47mmol) and K were added in that order₂CO₃(78mg, 0.56mmol) and KI (24mg, 0.14mmol), and reacted at reflux overnight. After the reaction is finished, cooling to room temperature, filtering, extracting filtrate for three times by using ethyl acetate and water, combining organic phases, washing by using saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column chromatography (D/M-60/1) gave C7-7(140mg, 41%) as a white solid.¹H NMR(400MHz，CDCl₃)δ8.68(s，1H，Ar-H)，7.88(d，J＝7.76Hz，1H，NH)，7.37-7.43(m，4H，Ar-H×4)，5.03-5.10(m，1H，CH)，4.86-4.89(m，1H，CH)，4.60-4.64(m，1H，CH)，3.61-3.68(m，2H，CH₂)，3.49(t，J＝4.20Hz，4H，CH₂×2)，3.12(s，1H，CH)，2.67-2.73(m，1H，CH_2-a)，2.34-2.56(m，11H，CH₃&CH₂×2&CH₂×2)，1.92-1.99(m，1H，CH_2-a)，1.27-1.55(m，22H，CH₃&CH₃×3&CH₂×5)，1.02(s，9H，CH₃×3)，；¹³C NMR(100MHz，CDCl₃)δ176.20，169.63，154.63，150.25，148.46，143.28，131.63，130.81，129.55，126.43，79.84，69.90，67.32，58.58，58.15，55.82，52.93，52.89，48.95，48.91，35.26，35.15，30.05，29.02，28.41，27.06，26.87，26.81，26.16，22.32，16.09.

b. Synthesis of intermediate C7-8

C7-7(0.11g, 0.15mmol) was dissolved in 10mL DCM, 1mL TFA was added, and the mixture was stirred at room temperature for 30 min. After the reaction is finished, removing the solvent under reduced pressure to obtain a light yellow liquid mixture C7-8, and carrying out the next reaction without separation;

(2) synthesis of R23

The same procedure and treatment as R13 were carried out using C7-8 and RSL3-S4 as starting materials to give R23 as a white solid (50%).

Spectral data of R23:¹H NMR(400MHz，DMSO-d⁶)δ10.97(s，1H，NH)，8.99(s，1H，Ar-H)，8.40(d，J＝7.48Hz，1H，NH)，7.37-7.57(m，8H，Ar-H&NH)，7.24-7.33(m，3H，Ar-H)，6.95-7.07(m，2H，Ar-H)，6.03(brs，1H，CH)，5.40(brs，1H，CH)，5.03(s，1H，CH)，4.88-4.94(m，1H，CH)，4.73(d，J＝14.2Hz，1H，CH_2a)，4.52(t，J＝8.08Hz，1H，CH)，4.42(d，J＝13.60Hz，1H，CH_2b)，4.28(s，1H，CH)，3.21-3.60(m，11H，CH₂×4&CH₃)，2.46(s，3H，CH₃)，2.20-2.42(m，8H，CH₂×4)，2.00-2.06(m，1H，CH)，1.76-1.83(m，1H，CH)，1.33-1.41(m，7H，CH₂×2&CH₃)，1.17-1.29(m，6H，CH₂×3)，0.92(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.19，171.21，169.08，168.21，163.33，151.93，148.21，145.21，144.34，136.83，134.68，134.47，131.57，130.14，129.42，129.29，127.47，126.78，126.21，121.79，119.37，118.49，111.87，104.02，69.21，67.31，66.61，66.07，65.48，58.79，58.15，57.10，56.64，56.47，53.25，48.15，43.80，37.94，35.33，29.29，27.34，27.23，27.05，26.58，23.95，22.97，22.62，16.46.M.P.177-178℃；HRMS(ESI+)：m/z calculated for C₅₆H₇₂ClN₈O₇S(M+H)₊：1035.4933；found 1035.4951.

examples 24 to 28 Synthesis of GPX4 protein degradation Agents R24 to 28

Examples 24 to 28 are different from example 23 in the value of n as a starting material in the preparation of a compound having a structure represented by the formula C-2.

Spectral data of R24:¹H NMR(400MHz，DMSO-d⁶)δ10.98(s，1H，NH)，8.97(s，1H，Ar-H)，8.40(brs，1H，NH)，7.35-7.56(m，8H，Ar-H&NH)，7.20-7.31(m，3H，Ar-H)，6.94-7.05(m，2H，Ar-H)，6.02(brs，1H，CH)，5.39(brs，1H，CH)，5.04(s，1H，CH)，4.87-4.94(m，1H，CH)，4.72(d，J＝13.84Hz，1H，CH_2a)，4.52(t，J＝8.04Hz，1H，CH)，4.40(d，J＝13.92Hz，1H，CH_2b)，4.27(s，1H，CH)，3.17-3.57(m，11H，CH₂×4&CH₃)，2.44(s，3H，CH₃)，2.20-2.40(m，8H，CH₂×4)，2.01-2.05(m，1H，CH)，1.75-1.82(m，1H，CH)，1.34-1.40(m，7H，CH₂×2&CH₃)，1.20-1.27(m，10H，CH₂×5)，0.92(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ171.76，170.75，168.69，167.82，163.22，151.53，147.81，144.79，143.98，136.43，134.24，134.08，131.65，131.18，129.75，128.90，127.08，126.40，125.82，121.36，118.96，118.09，111.47，103.62，68.82，67.47，66.91，66.16，65.66，58.40，57.75，56.69，56.31，56.08，52.85，47.77，43.44，38.16，34.93，29.87，28.99，28.44，26.95，26.80，26.61，26.22，23.32，22.58，22.25，16.07.M.P.118-119℃；HRMS(ESI+)：m/z calculated for C₅₈H₇₆ClN₈O₇S(M+H)₊：1063.5246；found 1063.5265.

spectral data of R25:¹H NMR(400MHz，DMSO-d⁶)δ10.97(s，1H，NH)，8.99(s，1H，Ar-H)，8.41(brs，1H，NH)，7.38-7.49(m，8H，Ar-H&NH)，7.24-7.32(m，3H，Ar-H)，6.96-7.05(m，2H，Ar-H)，6.03(brs，1H，CH)，5.40(brs，1H，CH)，5.05(s，1H，CH)，4.90-4.93(m，1H，CH)，4.73(d，J＝9.36Hz，1H，CH_2a)，4.53(t，J＝5.32Hz，1H，CH)，4.42(d，J＝9.24Hz，1H，CH_2b)，4.29(s，1H，CH)，3.24-3.59(m，11H，CH₂×4&CH₃)，2.46(s，3H，CH₃)，2.26-2.40(m，8H，CH₂×4)，2.02-2.07(m，1H，CH)，1.78-1.83(m，1H，CH)，1.38-1.42(m，7H，CH₂×2&CH₃)，1.23-1.25(m，14H，CH₂×7)，0.94(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.15，171.16，169.09，168.22，162.45，151.94，148.21，145.17，144.35，136.82，134.65，134.47，131.57，130.15，129.43，129.30，127.48，126.79，126.21，121.78，119.37，118.50，111.87，104.03，69.22，67.30，66.57，66.07，61.73，58.80，58.14，57.09，56.70，56.49，53.25，48.16，43.83，37.97，35.32，30.27，29.56，29.45，29.34，29.05，27.36，27.22，27.00，26.61，23.94，22.97，22.64，16.46；M.P.94-95℃；HRMS(ESI+)：m/z calculated for C₆₀H₈₀ClN₈O₇S(M+H)₊：1091.5559；found 1091.5547.

spectral data of R26:¹H NMR(400MHz，DMSO-d⁶)δ10.99(s，1H，NH)，8.99(s，1H，Ar-H)，8.43(brs，1H，NH)，7.38-7.49(m，8H，Ar-H&NH)，7.24-7.34(m，3H，Ar-H)，6.96-7.07(m，2H，Ar-H)，6.04(brs，1H，CH)，5.41(brs，1H，CH)，5.07(s，1H，CH)，4.90-4.95(m，1H，CH)，4.74(d，J＝9.32Hz，1H，CH_2a)，4.54(t，J＝4.80Hz，1H，CH)，4.43(d，J＝9.24Hz，1H，CH_2b)，4.30(s，1H，CH)，3.22-3.59(m，11H，CH₂×4&CH₃)，2.47(s，3H，CH₃)，2.21-2.42(m，8H，CH₂×4)，2.03-2.09(m，1H，CH)，1.79-1.83(m，1H，CH)，1.38-1.43(m，7H，CH₂×2&CH₃)，1.22-1.26(m，16H，CH₂×8)，0.95(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.15，171.12，169.09，168.22，162.45，151.94，148.21，145.17，144.37，136.83，134.63，134.47，131.57，130.16，129.43，129.30，127.48，126.79，126.22，121.77，119.37，118.49，111.87，104.02，69.22，67.30，66.50，66.06，61.73，58.89，58.12，57.09，56.75，56.48，53.25，48.17，43.83，37.99，35.32，30.27，29.58，29.51，29.47，29.44，29.34，27.35，27.17，26.99，26.57，23.94，22.97，22.65，16.46；M.P.105-106℃；HRMS(ESI+)：m/z calculated for C₆₁H₈₂ClN₈O₇S(M+H)₊：1105.5716；found 1105.5710.

spectral data of R27:¹H NMR(400MHz，DMSO-d⁶)δ10.96(s，1H，NH)，8.99(s，1H，Ar-H)，8.40(brs，1H，NH)，7.37-7.51(m，8H，Ar-H&NH)，7.24-7.28(m，3H，Ar-H)，6.97-7.05(m，2H，Ar-H)，6.03(brs，1H，CH)，5.40(brs，1H，CH)，5.03(s，1H，CH)，4.90-4.95(m，1H，CH)，4.73(d，J＝9.32Hz，1H，CH_2a)，4.54(t，J＝5.32Hz，1H，CH)，4.42(d，J＝9.24Hz，1H，CH_2b)，4.29(s，1H，CH)，3.19-3.59(m，11H，CH₂×4&CH₃)，2.47(s，3H，CH₃)，2.25-2.42(m，8H，CH₂×4)，2.03(br，1H，CH)，1.79-1.83(m，1H，CH)，1.35-1.41(m，7H，CH₂×2&CH₃)，1.22-1.26(m，18H，CH₂×9)，0.93(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.15，171.19，169.08，168.22，164.93，151.94，148.22，145.20，144.35，136.83，134.67，134.47，131.57，130.15，129.30，128.51，127.48，126.79，126.22，121.78，119.37，118.50，111.87，104.03，69.22，67.30，66.60，66.07，61.18，58.80，58.16，57.09，56.66，56.49，53.25，48.16，43.83，37.96，35.33，30.39，29.58，29.51，29.49，29.44，29.35，29.17，27.36，27.24，27.00，26.62，23.94，22.98，22.63，16.46；M.P.101-102℃；HRMS(ESI+)：m/z calculated for C₆₂H₈₄ClN₈O₇S(M+H)₊：1119.5872；found 1119.5884.

spectral data of R28:¹H NMR(400MHz，DMSO-d⁶)δ10.99(s，1H，NH)，8.99(s，1H，Ar-H)，8.42(brs，1H，NH)，7.37-7.50(m，8H，Ar-H&NH)，7.23-7.33(m，3H，Ar-H)，6.96-7.06(m，2H，Ar-H)，6.03(brs，1H，CH)，5.40(brs，1H，CH)，5.07(s，1H，CH)，4.91-4.93(m，1H，CH)，4.73(d，J＝9.40Hz，1H，CH_2a)，4.54(t，J＝5.08Hz，1H，CH)，4.42(d，J＝9.32Hz，1H，CH_2b)，4.29(brs，1H，CH)，3.43-3.56(m，11H，CH₂×4&CH₃)，2.46(s，3H，CH₃)，2.24-2.39(m，8H，CH₂×4)，2.05(br，1H，CH)，1.78-1.82(m，1H，CH)，1.38-1.41(m，7H，CH₂×2&CH₃)，1.23-1.25(m，20H，CH₂×10)，0.94(s，9H，CH₃×3)；¹³CNMR(400MHz，DMSO-d⁶)δ172.15，171.03，169.09，168.21，164.94，151.94，148.21，145.16，144.36，136.81，134.61，134.47，131.56，130.16，129.42，129.30，127.48，126.79，126.21，121.76，119.36，118.49，111.86，104.02，69.21，67.29，66.45，66.05，60.45，58.80，58.09，57.09，56.72，56.47，53.24，48.16，42.28，37.99，35.31，30.26，29.53，29.49，29.42，29.31，29.16，29.12，28.81，27.33，27.14，26.98，26.54，23.94，22.96，22.64，16.46；M.P.94-95℃；HRMS(ESI+)：m/z calculated for C₆₃H₈₆ClN₈O₇S(M+H)₊：1133.6029；found 1133.6030.

example 29 Synthesis of GPX4 protein degradation agent R29

a. Synthesis of intermediate C9-9

C9-8(0.72g, 1.09mmol) was dissolved in 10mL CH3CN, followed by the addition of 1-Boc-4-bromopiperidine (0.32g, 1.20mmol), K₂CO₃(0.25g, 1.79mmol) and KI (55mg, 0.33mmol) were reacted for 2h under reflux. After the reaction is finished, cooling to room temperature, filtering, extracting filtrate for three times by using ethyl acetate and water, combining organic phases, washing by using saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Separating by column chromatography (D/M: 60/1) to obtain white solid C9-9.

b. Synthesis of intermediate C9-10

C9-9(0.27g, 0.37mmol) was dissolved in 10mL DCM, 1mL TFA was added, and the mixture was stirred at room temperature for 30 min. After the reaction was completed, the solvent was removed under reduced pressure to obtain a pale yellow liquid mixture C9-10, which was subjected to the next reaction without separation.

Synthesis of GPX4 degradation agent R29

RSL3-S4(86mg, 0.18mmol) was dissolved in 8mL DMF and then C9-10(80mg, 0.18mmol), HATU (96mg, 0.25mmol) and DIPEA (140mg, 1.08mmol) were added sequentially and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was extracted three times with water and ethyl acetate, and the organic phases were combined, andwashed with saturated NaCl solution and anhydrous Na₂SO₄And (5) drying. Column chromatography (D/M-50/1) gave R29 as a white solid.

Example 30 Synthesis of protein degradation agent R30 of GPX4

a. Synthesis of intermediate RSL3-B1

D-Tryptophan methyl ester hydrochloride (2.57g, 11.75mmol) was dissolved in 15mL DCM, TEA (1.32g, 13.08mmol) was added, and the mixture was stirred at room temperature for 1 hour. The filtrate was filtered, dried by spinning, dissolved in 30mL of DCM, and p-tolualdehyde (1.28g, 10.68mmol), TFA (61mg, 0.53mmol) and

after refluxing for 1 hour, TFA (1.83g, 16.02mmol) was added again, and the reaction was refluxed overnight. After the reaction is finished, the reaction product is cooled to room temperature, quenched by 30 percent NaOH solution, separated by organic phase, washed by saturated NaCl solution for three times and anhydrous Na₂SO₄And (5) drying. Column separation (D/M ═ 80/1) gave a white solid, giving the compound identified as RSL 3-B1.

b. Synthesis of intermediate RSL3-B2

RSL3-B1(1.00g, 3.12mmol) was charged in 20mL CCl4, followed by NBS (0.36g, 6.24mmol) and reacted at reflux for 2 hours. After the reaction was complete, it was cooled to room temperature, quenched by the addition of 15mL of water, the organic phase separated, washed three times with saturated NaCl solution and dried over anhydrous Na2SO 4. Column separation (D/M ═ 80/1) gave a white solid, giving the compound identified as RSL 3-B2.

c. Synthesis of intermediate RSL3-B3

RSL3-B2(0.32g, 0.80mmol) was dissolved with 8mL dry DCM and NaHCO was added₃(74mg, 0.88mmol) and chloroacetyl chloride (0.11g, 0.96mmol) was added portionwise while cooling on ice and reacted at room temperature for 7 h. After the reaction, the reaction solution was quenched with water, extracted with ethyl acetate three times, and the organic phase was washed with saturated NaCl solution and anhydrous Na₂8O₄And (5) drying. Removing part of solvent under reduced pressure to precipitate a large amount of white solid, filtering, washing filter cake with a small amount of diethyl ether, and drying to obtain white solid RSL 3-B3.

Synthesis of GPX4 degradation agent R30

C13-8(80mg, 0.18mmol) was dissolved in 5mL CH3CN, and then RSL3-B3(86g, 0.18mmol), K were added in sequence₂CO₃(25mg, 0.18mmol 1) and KI (5.5mg, 0.033mmol) were refluxed for 2 h. After the reaction is finished, cooling to room temperature, filtering, extracting filtrate for three times by using ethyl acetate and water, combining organic phases, washing by using saturated NaCl solution and anhydrous Na in sequence₂SO₄And (5) drying. Column chromatography (D/M-60/1) gave R30 as a white solid.

Performance test 1 degradation agent induces degradation of GPX4 protein in tumor cells

Through Western Blot test, the invention detects the tumor cell GPX4 protein degradation capability of the designed and synthesized degradation agent molecule. Firstly, degradation agents R1-R12 with polyethylene glycol (PEG) structures as connecting groups are detected. The results are shown in FIG. 1, where a is the result of GPX4 degradation of R1-R8 at 10. mu.M in FIG. 1; b is the cell state of HT1080 cells treated by 10 mu M R9-R12 for 24 h; c is the GPX4 degradation result of R1-R8 at 1 mu M; d is the result of GPX4 degradation of R9-R12 at 1. mu.M. FIG. 1 shows the levels of GPX4 protein after 24 hours of action of HT1080 cells cultured in 6-well plates with the addition of 10. mu.M and 1. mu.M of different degradants. Wherein R5 and R10 have the most obvious effect on GPX4 protein degradation. In fig. 1, b shows that after 10 μ M of different degradation agents are added into HT1080 cells and act for 24 hours, the cells die obviously, which proves that the degradation agents have obvious anti-tumor effect.

The Western Blot test is carried out on synthesized GPX4 degrading agents R13-R28 of carbon chains. In HT1080 cells, the administration concentration is 0.1 mu M, and the Western Blot result after different degradants are respectively administered for 24 hours shows that GPX4 protein is obviously degraded, as shown in figure 2, a-f are the degradation results of GPX4 of different degradants in HT1080 cells; g is the degradation result of GPX4 of degrading agents R25-R28 in MCF7 cells. It can be seen that the degradation agent was able to efficiently degrade GPX4 protein in HT1080 cells, fig. 2g) and fig. 3d) show that R16 and R25-R28 were able to efficiently degrade GPX4 protein in MCF-7 cells.

The invention detects the degradation condition of GPX4 by R27 after adding R27 with different concentrations (10 mu M is diluted at the beginning) into HT1080 and Calu-1 cells for action, and detects the degradation condition of GPX4 protein by degradation agent which acts in HT1080, Calu-1 and MCF7 cells for different administration time (0.5-24h), as shown in figure 3, a) in figure 3 is a concentration gradient degradation test of GPX4 protein in HT1080 cells by degradation agent R27; b) performing a degradation test on the concentration gradient of the GPX4 protein in Calu-1 cells by using a degradation agent R27; c) is the degradation of R16 at different time points for GPX4 in HT1080 cells; d) is the degradation of GPX4 in MCF7 cells at different time points for R16; e) is the degradation of R27 at different time points for GPX4 in HT1080 cells; f) is the degradation of GPX4 in Calu-1 cells at different time points for R27. FIGS. 3a) and 3b) the results show that R27 has a concentration-dependent profile for GPX4 protein degradation in HT1080 cells and Calu-1 cells, with DC50 at 23.26nM and 136 nM. At the same time, e) in fig. 2 shows that R16 also shows a concentration-dependent effect of GPX4 degradation after 24 hours of action in HT1080 cells. The results of c), d), e), f) in FIG. 3 show that the degradants are time-dependent on the degradation of GPX4 in HT1080, Calu-1 and MCF7 cells. The results prove that the degradation agent has obvious GPX4 protein degradation effect in HT1080, Calu-1 and MCF7 cells.

Performance test 2 degradation agent R27 degrades GPX4 protein through ubiquitin-proteasome pathway

The method uses a GPX4 inhibitor ML210, a proteasome inhibitor MG132 and a ubiquitin activator E1 inhibitor PYR41 to pre-incubate cells for 2 hours respectively, then uses R27 to treat the cells, and finally detects the change result of a GPX4 protein band through WB, wherein the result is shown in figure 4, and a) in figure 4 is a competition test of the inhibitor ML210 on the degradation of GPX4 by R27; b) a test for verifying the mechanism of inducing GPX4 protein degradation for R27. As can be seen in a) of fig. 4, with increasing ML210 pretreatment concentration, degradation of GPX4 protein by R27 was gradually competitively inhibited. As can be seen from b) in fig. 4, the degradation of GPX4 protein by R27 can be significantly blocked by MG132 and PYR41, indicating that R27 induces the degradation of GPX4 protein through ubiquitin-proteasome pathway.

Performance test 3 tumor cell proliferation inhibition experiment

Results of tumor cell proliferation experiments fig. 5 shows, in fig. 5 a) is toxicity test of R27 on HEK293T cells; b) is a proliferation inhibition test of R27 on HT1080 cells, c) is a proliferation inhibition test of R27 on Calu-1 cells; d) the method is a proliferation inhibition test of degrading agents R13-R17 on HT1080 cells; e) the reagent is a proliferation inhibition test of degrading agents R21, R23-R26 and R28 on HT1080 cells.

(1) Toxicity test of degradation agent on HEK293T cells

The invention selects human embryonic kidney cells (HEK293T) to detect the cell proliferation inhibition effect of the degradation agent on normal cells within 24 hours by an MTT method, and simultaneously uses GPX4 covalent inhibitors RSL3 and ML210 as positive controls. The degradation agent R27 showed similar cytotoxicity to the inhibitor, and the results are shown in a) of fig. 5.

(2) Inhibiting effect of degradation agent on tumor cell proliferation

The present invention examined the cell proliferation inhibitory effect of the degradant on human fibrosarcoma cells (HT1080) and human lung cancer cells (Calu-1) within 24 hours, and used the GPX4 inhibitors RSL3 and ML210 as controls. Taking R27 as an example, as shown by b) and c) in FIG. 5, the cell proliferation inhibition effect of R27 on the two cell lines is obviously better than that of RSL3 and ML210, wherein the IC50 in HT1080 cells is 58nM, and the IC50 in Calu-1 cells is 25 nM. As can be seen from d), e) in fig. 5 and b in fig. 1, other degradants also showed significant antitumor effects.

Performance test 4

(1) Liproxstatin-1 blocks R27-induced iron death in tumor cells

In order to verify that the degradation agent plays a role in resisting tumor proliferation through an iron wire apoptosis pathway, the invention uses the iron death inhibitor Liproxstin-1 with different concentrations (the concentrations are 0, 0.006859, 0.020576, 0.061782, 0.185185, 0.555556, 1.666667 and 5 mu M respectively) to pre-incubate HT1080 cells for 2 hours, then adds the degradation agent R27(R27 final concentration is 0.1 mu M), and finally detects the cell viability after 24 hours, and the result is shown in figure 6, wherein a in figure 6 shows that the iron death inhibitor Liproxstatin-1 inhibits R27-induced iron death of HT1080 cells; b is an assay for R27 induction of ROS production in HT1080 cells.

As can be seen from a in FIG. 6, the treatment group to which the iron death inhibitor Lip-1 was added was able to significantly inhibit cell death relative to the treatment group to which only the degrading agent R27 was added (second group from left), and the cell survival rate increased with increasing Lip-1 concentration within a certain range, indicating that the inhibition of HT1080 cells by R27 occurred via the iron death pathway.

(2) R27 induces ROS production in HT1080 cells

R27 severely impairs the ability of tumor cells to eliminate lipid hydroperoxides by degrading GPX4 protein, ultimately leading to the occurrence of iron death. Therefore we used C11-BODIPY^581/591The probe monitored the level of ROS production by tumor cells following administration of the degradant R27 at various time points, and the results are shown in fig. 6 b.

As can be seen from b in FIG. 6, the sharp increase in fluorescence intensity was detected 2 hours after the administration of the degrading agent R27(0.1AM), indicating that ROS were produced in large quantities at this time, and indirectly indicating that GPX4 protein had begun to be bound and degraded by R27 2 hours before the administration, cell ROS production reached a maximum value around 3 to 4 hours, and cell ROS content gradually decreased after 4 hours, indicating that cells had begun to die in large quantities at this time; the trend of inhibitor RSL3(0.1 μ M) to induce ROS production by HT1080 cells was relatively mild relative to the degradants, and ROS production was significantly lower than in the degradant-treated group; in addition, we performed another set of control experiments simultaneously, i.e., cells were previously incubated with 20nM of the iron death inhibitor Lip-1 for 2 hours prior to each time point of R27 administration, to verify the blocking effect of Lip-1 on R27-induced cellular ROS production. The results show that the ROS production of the R27+ Lip-1 treated group is obviously reduced (especially significant in 2-6 h) compared with that of the R27 treated group, so that the ROS production and iron death of the tumor cells induced by R27 can be blocked by the iron death inhibitor Lip-1, namely the GPX4 degrading agent R27 can induce the iron death of the tumor cells.

Performance test 5 degradation agent induces GPX4 protein degradation in drug-resistant tumor cells and inhibits proliferation of drug-resistant tumor cells

The invention constructs a gefitinib-resistant human non-small cell lung cancer cell (H1650) strain, which is used for researching the degradation effect of GPX4 degradation agents R17 and R27 on GPX4 protein in drug-resistant tumor cells. Wild type (H1650) and drug-resistant type (H1650R) human NSCLC cells were treated with different concentrations (0.3, 0.1, 0.03, 0.01. mu.M) of R17 and R27 for 24 hours, respectively, and then the GPX4 protein level of the cells of the corresponding experimental groups was examined by Western blot assay.

Protein degradation of R17 and R27 in wild-type and drug-resistant H1650 cells is shown in fig. 7, a in fig. 7 is that different concentrations of R17 induce GPX4 degradation in H1650 cells; b is that different concentrations of R17 induced GPX4 degradation in H1650R cells; c is the induction of GPX4 degradation in H1650 cells at different concentrations of R27; d is the induction of GPX4 degradation in H1650R cells by different concentrations of R27; e is the proliferation inhibition of the inhibitor RSL3 and the degradation agent R27 on wild type and drug-resistant H1650 cells respectively. It can be seen that R27 was able to significantly induce GPX4 degradation in wild-type and drug-resistant H1650 cells 24 hours after administration, and was concentration-dependent. Wherein GPX4 is almost completely degraded at a concentration of R27 of 0.01. mu.M in H1650 cells; for H1650R cells, the concentration of R27 is 0.03 mu M, and GPX4 can be degraded significantly; r17 can degrade most of GPX4 protein in H1650 cells at 0.01 mu M, and can also degrade GPX4 protein in H1650R cells in a concentration-dependent manner; compared with RSL3, the degradation agent R27 can better inhibit the proliferation of wild type and drug-resistant H1650 cells, and the inhibition effect on the drug-resistant H1650R cells is better than that of the wild type H1650 cells. The experimental result shows that the GPX4 degrading agent can degrade GPX4 protein in wild type and drug-resistant tumor cells simultaneously, and the GPX4 protein degraded by the GPX4 degrading agent can well induce the death of drug-resistant tumor cells.

Performance test pharmacodynamic study of 6GPX4 degradation agent on xenograft tumor mice

The invention constructs a mouse model of xenograft human fibrosarcoma (HT1080) and is used for evaluating the in vivo pharmacodynamic action of the GPX4 degrading agent.

(1) Degradation effect of R27 on GPX4 protein of tumor tissue

With reference to the administration pattern of the GPX4 inhibitor RSL3, R27(100mg/kg) was subcutaneously administered peritumorally, tumor tissues were taken at designated time points, and proteins were extracted to perform Western blot assay, and the results are shown in fig. 8. Degradation of the GPX4 protein in the tumor tissue occurred 3 hours after R27 administration and was time-dependent within 12 hours, whereas the color of the band was deepened at the later time points of 24 hours and 48 hours, indicating that the degradation agent may have been completely consumed after 12 hours, while the content of GPX4 protein increased by biological re-synthesis. The results show that the degradation agent R27 still has obvious GPX4 protein degradation effect in vivo experiments.

(2) Effect of R27 on mouse tumor volume

We used degradant R27 (three concentrations of 50mg/kg, 100mg/kg and 200 mg/kg), inhibitor RSL3(100mg/kg) and a blank solvent control to respectively subcutaneously administer the tumor in mice once every four days, and measure the tumor size and the body weight of mice, thereby comparing the effects on the tumor volume and body weight of mice after multiple administrations of different kinds of drugs. The results are shown in FIG. 9. In FIG. 9, a is the change trend of the tumor volume of the mice, and b is the change trend of the body weight of the mice.

As can be seen from fig. 9, the body weight of the mice in each group decreased and the tumor volume showed a tendency to increase after four times of administration, wherein the control group showed the most significant increase in the 50mg/kg R27-treated group, the 100mg/kg R27 and 100mg/kg RSL 3-treated groups showed more gradual and almost consistent increase in the tumor volume, and the inhibition of the increase in the tumor volume was further increased after 12 days of administration in the 200mg/kg R27-administered group. Since the molecular weight of R27 (m.w.: 1119.91) is about 2.5 times the molecular weight of RSL3 (m.w.: 440.88), these data indirectly indicate that the tumor-inhibiting effect of the degradant R27 is about 2.5 times that of the inhibitor RSL3 when administered at the same molar amount.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A GPX4 protein degradation agent has a structure shown in formula 1:

in the formula 1, A₁Is of the formula A₁₁～A₁₂Any one of the substituents shown:

A₁₁and A₁₂M is 1-8;

A₂is of the formula A₂₁～A₂₂Any one of the substituents shown:

A₂₁wherein X is CH₂Or CO; a. the₂₂In which Y is CH₃Or H.

2. A GPX4 protein degradation agent has a structure shown in formula 2, formula 3, formula 4, formula 5, formula 6 or formula 7:

in the formula 2, m is 1-8;

in the formula 3, m is 1-8;

in the formula 4, m is 1-8;

in the formula 5, n is 5-20, and o is 0-3;

in the formula 6, n is 5-20, and o is 0-3;

in the formula 7, n is 5-20, and o is 0-3;

in the formula 8, n is 5 to 20, and o is 0 to 3.

3. A method of making the GPX4 protein degrading agent of claim 2, comprising the steps of:

firstly, carrying out Click reaction on an E3 ligand compound substituted by an azido polyethylene glycol chain and a first GPX4 ligand to obtain a GPX4 protein degradation agent;

the first GPX4 ligand has the structure shown in formula B:

condensation reaction of E3 ligand compound containing piperazine ring carbon chain substitution and the second GPX4 ligand to obtain GPX4 protein degrading agent;

or the E3 ligand compound containing piperazine ring carbon chain substitution and a third GPX4 ligand are subjected to nucleophilic substitution reaction to obtain a GPX4 protein degradation agent;

the E3 ligand compound containing piperazine ring carbon chain substitution has a structure shown in a formula C-1, a formula C-2, a formula C-3 or a formula C-4:

the second GPX4 ligand has the structure shown in formula D:

the third GPX4 ligand has the structure shown in formula E:

4. the method according to claim 3, wherein the azido-polyethylene glycol chain-substituted E3 ligand compound having the structure represented by the formula A-1 is prepared by a method comprising the steps of:

5. the method of claim 3, wherein the first GPX4 ligand having the structure of formula B comprises the following steps:

6. the method according to claim 3, wherein the piperazine ring carbon chain-substituted E3 ligand compound having the structure represented by formula C-1 is prepared by a method comprising the steps of:

carrying out deprotection reaction on the compound with the structure shown as the formula L to obtain an E3 ligand compound containing piperazine ring carbon chain substitution with the structure shown as the formula C-1;

a compound having a structure represented by formula n and H₂Carrying out a hydrogenation reactionTo obtain a compound with a structure shown in a formula o;

7. The method of claim 3, wherein the second GPX4 ligand having the structure of formula D comprises the steps of:

8. the method of claim 3, wherein the third GPX4 ligand having the structure of formula E comprises the following steps:

9. the GPX4 protein degrading agent of claim 1 or 2 or the GPX4 protein degrading agent prepared by the preparation method of claims 3-8, and the application thereof in preparing anti-tumor drugs and drug-resistant tumor drugs.

10. An antitumor drug comprises a pharmaceutically active component and a pharmaceutical adjuvant; the active pharmaceutical ingredient is the GPX4 protein degrading agent of claim 1 or 2 or the GPX4 protein degrading agent prepared by the preparation method of claims 3-8.